Reprogramming cells

ABSTRACT

The present invention provides for methods, compositions, and kits for producing an induced pluripotent stem cell from a non-pluripotent mammalian cell using a 3′-phosphoinositide-dependent kinase-1 (PDK1) activator or a compound that promotes glycolytic metabolism as well as other small molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/069,730, filed Mar. 14, 2016, issued as U.S. Pat. No. 9,657,274,which is a divisional of U.S. application Ser. No. 13/896,259, filed May16, 2013, issued as U.S. Pat. No. 9,315,779, which is a continuation ofU.S. application Ser. No. 13/637,334, filed Sep. 25, 2012, which is aU.S. National Stage entry under 35 U.S.C. § 371 of PCT/US2011/030598,filed Mar. 30, 2011, which claims priority to U.S. ProvisionalApplication No. 61/319,494, filed Mar. 31, 2010, U.S. ProvisionalApplication No. 61/393,724, filed Oct. 15, 2010, and U.S. ProvisionalApplication No. 61/406,892, filed Oct. 26, 2010, the contents of each ofwhich is incorporated by reference in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file077103-1044239-004040US_SequenceListing.txt, created on Apr. 12, 2017,14,689 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Induced pluripotent stem cell (iPSC) technology, i.e. reprogrammingsomatic cells into pluripotent cells that closely resemble embryonicstem cells (ESCs) by introduction of defined factors, holds greatpotential in biomedical research and regenerative medicine (Takahashi,K., and Yamanaka, S., Cell 126, 663-676 (2006); Takahashi et al., Cell131, 861-872 (2007); Yu et al., Science 318, 1917-1920 (2007); Zhou etal., Cell Stem Cell 4, 381-384 (2009); Kim et al., Cell Stem Cell 4,472-476 (2009); Maherali, N., and Hochedlinger, K., Cell Stem Cell 3,595-605 (2009a); Daley et al., Cell Stem Cell 4, 200-201 (2009)).Various strategies have been developed to generate iPSCs with less or noexogenous genetic manipulations, which represent a major hurdle for iPSCapplications (Yamanaka et al., 2009; Saha, K., Jaenisch, R., Cell StemCell 5, 584-595 (2009)). Toward an ultimate goal of generating iPSCswith a defined small molecule cocktail that would offer significantadvantages over genetic manipulations or moredifficult-to-manufacture/use biologics, substantial efforts have beenmade in identifying chemical compounds that can functionally replaceexogenous reprogramming transcription factors (TFs) and/or enhancereprogramming efficiency and kinetics (Shi et al., Cell Stem Cell 2,525-528 (2008a); Shi et al., Cell Stem Cell 3, 568-574 (2008b); Huangfuet al., Nat Biotechnol 26, 795-797 (2008a); Huangfu et al., NatBiotechnol 26, 1269-1275 (2008b); Silva et al., Plos Bio 6, e253. doi:10.1371/journal.pbio. 0060253 (2008); Lyssiotis et al., PNAS 106,8912-8917 (2009); Ichida et al., Cell Stem Cell 5, 491-503 (2009);Maherali, N., Hochedlinger, K., Curr Biol 19, 1718-1723 (2009b); Estebanet al., Cell Stem Cell 6, 71-79 (2010); Feng et al., Cell Stem Cell 4,301-312 (2009)). However, further reducing the number of exogenous TFshas been extraordinarily challenging as (1) most reprogramming enablingor enhancing conditions (e.g., exploiting a specific cell type or usingsmall molecules) are context dependent, i.e., such specific conditions(e.g., a reprogramming small molecule) typically would be much lesseffective or even harmful in a different cell type with differentexogenous factors and used in a different window of treatment; and (2)high throughput screening is technically challenging when thereprogramming efficiency and speed further decrease exponentially due tofewer exogenous TFs used. To date, only neural stem cells (NSCs) thatendogenously express Sox2 and cMyc at a high level were shown to bereprogrammed to iPSCs by exogenous expression of only Oct4 (Kim et al.,Cell 136, 411-419 (2009a); Kim et al., Nature 461, 643-649 (2009b)).However, human fetal NSCs are rare and practically difficult to obtain(Nunes et al., Nat Med 9, 439-447 (2003)). Consequently, it would bebeneficial to develop chemical reprogramming conditions applicable toother more accessible and abundant somatic cells.

BRIEF SUMMARY OF THE INVENTION

The present invention provides for a method of inducing anon-pluripotent mammalian cell into an induced pluripotent stem cell. Insome embodiments, the method comprises contacting the non-pluripotentcell with a 3′-phosphoinositide-dependent kinase-1 (PDK1) activatorunder conditions sufficient to induce the cell to become a pluripotentstem cell. In some embodiments, the PDK1 activator is an allosteric PDK1activator, e.g., (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid(“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid(“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”).

In some embodiments, the method further comprises contacting thenon-pluripotent cell with a TGFβ receptor/ALK5 inhibitor, e.g., A-83-01.In some embodiments, the method further comprises contacting thenon-pluripotent cell with a MEK inhibitor, e.g., PD0325901. In someembodiments, the method further comprises contacting the non-pluripotentcell with a histone deacetylase (HDAC) inhibitor, e.g., sodium butyrate(NaB), or valproic acid (VPA).

In some embodiments, the method comprises contacting the non-pluripotentcell with a 3′-phosphoinositide-dependent kinase-1 (PDK1) activatorunder conditions sufficient to induce the cell to become a pluripotentstem cell. In some embodiments, the conditions comprise introducing atleast one exogenous transcription factor into the non-pluripotent cell.In some embodiments, the exogenous transcription factor comprises apolypeptide. In some embodiments, the exogenous transcription factorcomprises an Oct polypeptide. In some embodiments, the exogenoustranscription factor comprises a protein selected from the groupconsisting of an Oct polypeptide and a Klf polypeptide. In someembodiments, the exogenous transcription factor comprises a proteinselected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the condition comprises introducing at least two, three orfour exogenous transcription factors into the non-pluripotent cell,wherein the exogenous transcription factors each comprise a differentprotein selected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor is introduced byintroducing a polynucleotide into the non-pluripotent cell, wherein thepolynucleotide encodes the exogenous transcription factor, therebyexpressing the transcription factor(s) in the non-pluripotent cell. Insome embodiments, the exogenous transcription factor is introduced bycontacting the exogenous transcription factor to the non-pluripotentcell. In some embodiments, the exogenous transcription factor comprisesan amino acid sequence that enhances transport across cell membranes.

In some embodiments, the non-pluripotent cell is a human cell. In someembodiments, the PDK1 activator is present in a concentration sufficientto improve by at least 10% the efficiency of induction of thenon-pluripotent cell into an induced pluripotent stem cell, underconditions sufficient to induce conversion of the non-pluripotent cellinto the induced pluripotent stem cell.

In some embodiments, the method comprises contacting the non-pluripotentcell with a 3′-phosphoinositide-dependent kinase-1 (PDK1) activatorunder conditions sufficient to induce the cell to become a pluripotentstem cell. In some embodiments, the method comprises contacting thenon-pluripotent cell with a PDK1 activator in the absence of a MEKinhibitor, followed by contacting the non-pluripotent cell with a PDK1activator and a MEK inhibitor. In some embodiments, the method comprisescontacting the non-pluripotent cell with a PDK1 activator, a TGFβreceptor/ALK5 inhibitor, and a histone deacetylase (HDAC) inhibitor inthe absence of a MEK inhibitor, followed by contacting thenon-pluripotent cell with a PDK1 activator, a TGFβ receptor/ALK5inhibitor, a histone deacetylase (HDAC) inhibitor and a MEK inhibitor.

In some embodiments, the method further comprises purifying thepluripotent cells to generate a homogenous population of the pluripotentcells. In some embodiments, wherein a plurality of pluripotent stemcells are induced, the method further comprises purifying thepluripotent stem cells to generate a homogenous population ofpluripotent stem cells.

In another aspect, the present invention provides for a mixturecomprising: mammalian cells, a PDK1 activator, and one or more of (1) aTGFβ receptor/ALK5 inhibitor; (2) a MEK inhibitor; (3) a histonedeacetylase (HDAC) inhibitor; or (4) one or more exogenous transcriptionfactors selected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide.

In some embodiments, at least 99% of the cells in the mixture arenon-pluripotent cells. In some embodiments, essentially all of the cellsare non-pluripotent cells. In some embodiments, the cells are humancells. In some embodiments, the PDK1 activator is an allosteric PDK1activator, e.g., (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid(“PS48”), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid(“PS08”), 2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”). In someembodiments, the mixture further comprises a TGFβ receptor/ALK5inhibitor, e.g., A-83-01. In some embodiments, the mixture furthercomprises a MEK inhibitor, e.g., PD0325901. In some embodiments, themixture further comprises a histone deacetylase (HDAC) inhibitor, e.g.,sodium butyrate (NaB), or valproic acid (VPA).

In some embodiments, the mixture further comprises an exogenoustranscription factor selected from an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor comprises an amino acidsequence that enhances transport across cell membranes. In someembodiments, the PDK1 activator in the mixture is present in aconcentration sufficient to improve by at least 10% the efficiency ofinduction of non-pluripotent cells in the mixture into inducedpluripotent stem cells under conditions sufficient to induce conversionof the cells into induced pluripotent stem cells.

In still another aspect, the present invention provides for a kit forinducing pluripotency in a non-pluripotent mammalian cell, the kitcomprising a PDK1 activator, and one or more of (1) a TGFβ receptor/ALK5inhibitor; (2) a MEK inhibitor; (3) a histone deacetylase (HDAC)inhibitor; or (4) one or more transcription factors selected from thegroup consisting of an Oct polypeptide, a Klf polypeptide, a Mycpolypeptide, and a Sox polypeptide. In some embodiments, the PDK1activator is an allosteric PDK1 activator, e.g.,(Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (“PS48”),(Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (“PS08”),2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (“12Z”), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (“13Z”). In someembodiments, the kit further comprises a TGFβ receptor/ALK5 inhibitor,e.g., A-83-01. In some embodiments, the kit further comprises a MEKinhibitor, e.g., PD0325901. In some embodiments, the kit furthercomprises a histone deacetylase (HDAC) inhibitor, e.g., sodium butyrate(NaB), or valproic acid (VPA).

In some embodiments, the kit further comprises an exogenoustranscription factor selected from an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor comprises an amino acidsequence that enhances transport across cell membranes.

In yet another aspect, the present invention provides a method ofinducing a non-pluripotent mammalian cell into an induced pluripotentstem cell. In some embodiments, the method comprises contacting thenon-pluripotent cell with a compound that promotes glycolytic metabolismunder conditions sufficient to induce the cell to become a pluripotentstem cell, thereby inducing the non-pluripotent mammalian cell into aninduced pluripotent stem cell. In some embodiments, the compound thatpromotes glycolytic metabolism is a PDK1 activator. In some embodiments,the PDK1 activator is an allosteric PDK1 activator, e.g., PS48, PS08,12Z, or 13Z. In some embodiments, the compound that promotes glycolyticmetabolism is a glycolysis activator, e.g., fructose 2,6-bisphosphate.In some embodiments, the compound that promotes glycolytic metabolism isa substrate for glycolysis, e.g., fructose 6-phosphate. In someembodiments, the compound that promotes glycolytic metabolism is aglycolytic intermediate or its metabolic precursors, e.g., nicotinicacid, NADH, or fructose 6-phosphate. In some embodiments, the compoundthat promotes glycolytic metabolism is a glucose uptake transporteractivator. In some embodiments, the compound that promotes glycolyticmetabolism is a mitochondrial respiration modulator. In someembodiments, the mitochondrial respiration modulator is an oxidativephosphorylation inhibitor, e.g., 2,4-dinitrophenol, or 2-hydroxyglutaricacid. In some embodiments, the compound that promotes glycolyticmetabolism is a hypoxia-inducible factor activator, e.g.,N-oxalylglycine, or quercetin. In some embodiments, the method furthercomprises contacting the non-pluripotent cell with a TGFβ receptor/ALK5inhibitor, e.g., A-83-01. In some embodiments, the method furthercomprises contacting the non-pluripotent cell with a MEK inhibitor,e.g., PD0325901. In some embodiments, the method further comprisescontacting the non-pluripotent cell with a histone deacetylase (HDAC)inhibitor, e.g., sodium butyrate (NaB), or valproic acid (VPA).

In some embodiments, the method comprises contacting the non-pluripotentcell with a compound that promotes glycolytic metabolism underconditions sufficient to induce the cell to become a pluripotent stemcell. In some embodiments, the conditions comprise introducing at leastone exogenous transcription factor into the non-pluripotent cell. Insome embodiments, the exogenous transcription factor comprises apolypeptide. In some embodiments, the exogenous transcription factorcomprises an Oct polypeptide. In some embodiments, the exogenoustranscription factor comprises a protein selected from the groupconsisting of an Oct polypeptide and a Klf polypeptide. In someembodiments, the exogenous transcription factor comprises a proteinselected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the condition comprises introducing at least two, three orfour exogenous transcription factors into the non-pluripotent cell,wherein the exogenous transcription factors each comprise a differentprotein selected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor is introduced byintroducing a polynucleotide into the non-pluripotent cell, wherein thepolynucleotide encodes the exogenous transcription factor, therebyexpressing the transcription factor(s) in the non-pluripotent cell. Insome embodiments, the exogenous transcription factor is introduced bycontacting the exogenous transcription factor to the non-pluripotentcell. In some embodiments, the exogenous transcription factor comprisesan amino acid sequence that enhances transport across cell membranes.

In some embodiments, the non-pluripotent cell is a human cell. In someembodiments, the compound that promotes glycolytic metabolism is presentin a concentration sufficient to improve by at least 10% the efficiencyof induction of the non-pluripotent cell into an induced pluripotentstem cell, under conditions sufficient to induce conversion of thenon-pluripotent cell into the induced pluripotent stem cell.

In some embodiments, the method comprises contacting the non-pluripotentcell with a compound that promotes glycolytic metabolism underconditions sufficient to induce the cell to become a pluripotent stemcell. In some embodiments, the method comprises contacting thenon-pluripotent cell with a compound that promotes glycolytic metabolismand a MEK inhibitor. In some embodiments, the method comprisescontacting the non-pluripotent cell with a compound that promotesglycolytic metabolism in the absence of a MEK inhibitor, followed bycontacting the non-pluripotent cell with a compound that promotesglycolytic metabolism and a MEK inhibitor. In some embodiments, themethod comprises contacting the non-pluripotent cell with a compoundthat promotes glycolytic metabolism, a TGFβ receptor/ALK5 inhibitor, anda histone deacetylase (HDAC) inhibitor in the absence of a MEKinhibitor, followed by contacting the non-pluripotent cell with acompound that promotes glycolytic metabolism, a TGFβ receptor/ALK5inhibitor, a histone deacetylase (HDAC) inhibitor and a MEK inhibitor.

In some embodiments, the method further comprises purifying thepluripotent cells to generate a homogenous population of the pluripotentcells. In some embodiments, wherein a plurality of pluripotent stemcells are induced, the method further comprises purifying thepluripotent stem cells to generate a homogenous population ofpluripotent stem cells.

In still another aspect, the present invention provides for a mixturecomprising: mammalian cells, a compound that promotes glycolyticmetabolism, and one or more of (1) a TGFβ receptor/ALK5 inhibitor; (2) aMEK inhibitor; (3) a histone deacetylase (HDAC) inhibitor; or (4) one ormore exogenous polypeptides selected from the group consisting of an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide. In some embodiments, at least 99% of the cells in themixture are initially non-pluripotent cells. In some embodiments,essentially all of the cells are initially non-pluripotent cells. Insome embodiments, the cells are human cells. In some embodiments, thecompound that promotes glycolytic metabolism is a PDK1 activator. Insome embodiments, the PDK1 activator is an allosteric PDK1 activator,e.g., PS48, PS08, 12Z, or 13Z. In some embodiments, the compound thatpromotes glycolytic metabolism is a glycolysis activator, e.g., fructose2,6-bisphosphate. In some embodiments, the compound that promotesglycolytic metabolism is a substrate for glycolysis, e.g., fructose6-phosphate. In some embodiments, the compound that promotes glycolyticmetabolism is a glycolytic intermediate or its metabolic precursors,e.g., nicotinic acid, NADH, or fructose 6-phosphate. In someembodiments, the compound that promotes glycolytic metabolism is aglucose uptake transporter activator. In some embodiments, the compoundthat promotes glycolytic metabolism is a mitochondrial respirationmodulator. In some embodiments, the mitochondrial respiration modulatoris an oxidative phosphorylation inhibitor, e.g., 2,4-dinitrophenol, or2-hydroxyglutaric acid. In some embodiments, the compound that promotesglycolytic metabolism is a hypoxia-inducible factor activator, e.g.,N-oxalylglycine, or quercetin. In some embodiments, the mixture furthercomprises a TGFβ receptor/ALK5 inhibitor, e.g., A-83-01. In someembodiments, the mixture further comprises a MEK inhibitor, e.g.,PD0325901. In some embodiments, the mixture further comprises a histonedeacetylase (HDAC) inhibitor, e.g., sodium butyrate (NaB), or valproicacid (VPA).

In some embodiments, the exogenous transcription factor comprises anamino acid sequence that enhances transport across cell membranes. Insome embodiments, the compound that promotes glycolytic metabolism inthe mixture is present in a concentration sufficient to improve by atleast 10% the efficiency of induction of non-pluripotent cells in themixture into induced pluripotent stem cells under conditions sufficientto induce conversion of the cells into induced pluripotent stem cells.

In yet another aspect, the present invention provides for a kit forinducing pluripotency in a non-pluripotent mammalian cell, the kitcomprising a compound that promotes glycolytic metabolism, and one ormore of (1) a TGFβ receptor/ALK5 inhibitor; (2) a MEK inhibitor; (3) ahistone deacetylase (HDAC) inhibitor; or (4) one or more transcriptionfactors selected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide; or apolynucleotide encoding a transcription factor selected from an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide. In some embodiments, the compound that promotes glycolyticmetabolism is a PDK1 activator. In some embodiments, the PDK1 activatoris an allosteric PDK1 activator, e.g., PS48, PS08, 12Z, or 13Z. In someembodiments, the compound that promotes glycolytic metabolism is aglycolysis activator, e.g., fructose 2,6-bisphosphate. In someembodiments, the compound that promotes glycolytic metabolism is asubstrate for glycolysis, e.g., fructose 6-phosphate. In someembodiments, the compound that promotes glycolytic metabolism is aglycolytic intermediate or its metabolic precursors, e.g., nicotinicacid, NADH, or fructose 6-phosphate. In some embodiments, the compoundthat promotes glycolytic metabolism is a glucose uptake transporteractivator. In some embodiments, the compound that promotes glycolyticmetabolism is a mitochondrial respiration modulator. In someembodiments, the mitochondrial respiration modulator is an oxidativephosphorylation inhibitor, e.g., 2,4-dinitrophenol, or 2-hydroxyglutaricacid. In some embodiments, the compound that promotes glycolyticmetabolism is a hypoxia-inducible factor activator, e.g.,N-oxalylglycine, or quercetin. In some embodiments, the kit furthercomprises a TGFβ receptor/ALK5 inhibitor, e.g., A-83-01. In someembodiments, the kit further comprises a MEK inhibitor, e.g., PD0325901.In some embodiments, the kit further comprises a histone deacetylase(HDAC) inhibitor, e.g., sodium butyrate (NaB), or valproic acid (VPA).In some embodiments, the exogenous transcription factor comprises anamino acid sequence that enhances transport across cell membranes.

Definitions

An “Oct polypeptide” refers to any of the naturally-occurring members ofOctamer family of transcription factors, or variants thereof thatmaintain transcription factor activity, e.g., within at least 50%, 80%,or 90% activity compared to the closest related naturally occurringfamily member, or polypeptides comprising at least the DNA-bindingdomain of the naturally occurring family member, and can furthercomprise a transcriptional activation domain. Exemplary Oct polypeptidesinclude, Oct-1, Oct-2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11.e.g. Oct3/4 (referred to herein as “Oct4”) contains the POU domain, a150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2, anduric-86. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev. 11, 1207-1225(1997). In some embodiments, variants have at least 85%, 90%, or 95%amino acid sequence identity across their whole sequence compared to anaturally occurring Oct polypeptide family member such as those listedabove or such as listed in Genbank accession number NP_002692.2 (humanOct4) or NP_038661.1 (mouse Oct4). Oct polypeptides (e.g., Oct3/4) canbe from human, mouse, rat, bovine, porcine, or other animals. Generally,the same species of protein will be used with the species of cells beingmanipulated.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Kruppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Kruppel, or variants of thenaturally-occurring members that maintain transcription factor activity,similar e.g., within at least 50%, 80%, or 90% activity compared to theclosest related naturally occurring family member, or polypeptidescomprising at least the DNA-binding domain of the naturally occurringfamily member, and can further comprise a transcriptional activationdomain. See, Dang, D. T., Pevsner, J. & Yang, V. W. Cell Biol. 32,1103-1121 (2000). Exemplary Klf family members include, Klf1, Klf2,Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, Klf10, Klf11, Klf12, Klf13,Klf14, Klf15, Klf16, and Klf17. Klf2 and Klf-4 were found to be factorscapable of generating iPS cells in mice, and related genes Klf1 and Klf5did as well, although with reduced efficiency. See, Nakagawa, et al.,Nature Biotechnology 26:101-106 (2007). In some embodiments, variantshave at least 85%, 90%, or 95% amino acid sequence identity across theirwhole sequence compared to a naturally occurring Klf polypeptide familymember such as those listed above or such as listed in Genbank accessionnumber CAX16088 (mouse Klf4) or CAX14962 (human Klf4). Klf polypeptides(e.g., Klf1, Klf4, and Klf5) can be from human, mouse, rat, bovine,porcine, or other animals. Generally, the same species of protein willbe used with the species of cells being manipulated. To the extent a Klfpolypeptide is described herein, it can be replaced with anestrogen-related receptor beta (Essrb) polypeptide. Thus, it is intendedthat for each Klf polypeptide embodiment described herein, acorresponding embodiment using Essrb in the place of a Klf4 polypeptideis equally described.

A “Myc polypeptide” refers any of the naturally-occurring members of theMyc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. CellBiol. 6:635-645 (2005)), or variants thereof that maintain transcriptionfactor activity, e.g., within at least 50%, 80%, or 90% activitycompared to the closest related naturally occurring family member, orpolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member, and can further comprise a transcriptionalactivation domain. Exemplary Myc polypeptides include, e.g., c-Myc,N-Myc and L-Myc. In some embodiments, variants have at least 85%, 90%,or 95% amino acid sequence identity across their whole sequence comparedto a naturally occurring Myc polypeptide family member, such as thoselisted above or such as listed in Genbank accession number CAA25015(human Myc). Myc polypeptides (e.g., c-Myc) can be from human, mouse,rat, bovine, porcine, or other animals. Generally, the same species ofprotein will be used with the species of cells being manipulated. To theextent a Myc polypeptide is described herein, it can be replaced with aWnt polypeptide, e.g., Wnt 3A (e.g., NP_149122.1), or agent thatstimulates the Wnt signaling pathway, e.g., a glycogen synthase kinasealpha or beta inhibitor. Thus, it is intended that for each Mycpolypeptide embodiment described herein, a corresponding embodimentusing a Wnt polypeptide or agent that stimulates the Wnt signalingpathway in the place of a Myc polypeptide is equally described.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain transcription factor activity, e.g., within atleast 50%, 80%, or 90% activity compared to the closest relatednaturally occurring family member or polypeptides comprising at leastthe DNA-binding domain of the naturally occurring family member, and canfurther comprise a transcriptional activation domain. See, e.g., Dang,D. T., et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000).Exemplary Sox polypeptides include, e.g., Sox1, Sox-2, Sox3, Sox4, Sox5,Sox6, Sox7, Sox8, Sox9, Sox10, Sox11, Sox12, Sox13, Sox14, Sox15, Sox17,Sox18, Sox-21, and Sox30. Sox1 has been shown to yield iPS cells with asimilar efficiency as Sox2, and genes Sox3, Sox15, and Sox18 have alsobeen shown to generate iPS cells, although with somewhat less efficiencythan Sox2. See, Nakagawa, et al., Nature Biotechnology 26:101-106(2007). In some embodiments, variants have at least 85%, 90%, or 95%amino acid sequence identity across their whole sequence compared to anaturally occurring Sox polypeptide family member such as those listedabove or such as listed in Genbank accession number CAA83435 (humanSox2). Sox polypeptides (e.g., Sox1, Sox2, Sox3, Sox15, or Sox18) can befrom human, mouse, rat, bovine, porcine, or other animals. Generally,the same species of protein will be used with the species of cells beingmanipulated.

An “exogenous transcription factor,” as used herein, refers to atranscription factor that is not naturally (i.e., endogenously)expressed in a cell of interest. Thus, an exogenous transcription factorcan be expressed from an introduced expression cassette (e.g., undercontrol of a promoter other than a native transcription factor promoter)or can be introduced as a protein from outside the cell. In someembodiments, the exogenous transcription factor comprises an Octpolypeptide (e.g., Oct4), a Klf polypeptide (e.g., Klf4), a Mycpolypeptide (e.g., c-Myc), or a Sox polypeptide (e.g., Sox2).

“H3K9” refers to histone H3 lysine 9. H3K9 modifications associated withgene activity include H3K9 acetylation and H3K9 modifications associatedwith heterochromatin, include H3K9 di-methylation or tri-methylation.See, e.g., Kubicek, et al., Mol. Cell 473-481 (2007). “H3K4” refers tohistone H3 lysine 4. See, e.g., Ruthenburg et al., Mol. Cell 25:15-30(2007).

The term “pluripotent” or “pluripotency” refers to cells with theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm).Pluripotent stem cells can contribute to many or all tissues of aprenatal, postnatal or adult animal. A standard art-accepted test, suchas the ability to form a teratoma in 8-12 week old SCID mice, can beused to establish the pluripotency of a cell population, howeveridentification of various pluripotent stem cell characteristics can alsobe used to detect pluripotent cells.

“Pluripotent stem cell characteristics” refer to characteristics of acell that distinguish pluripotent stem cells from other cells. Theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm) is apluripotent stem cell characteristic. Expression or non-expression ofcertain combinations of molecular markers are also pluripotent stem cellcharacteristics. For example, human pluripotent stem cells express atleast one, two, or three, and optionally all, of the markers from thefollowing non-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,TRA-2-49/6E, ALP, Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cellmorphologies associated with pluripotent stem cells are also pluripotentstem cell characteristics.

A “recombinant” polynucleotide is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacids.

“Expression cassette” refers to a polynucleotide comprising a promoteror other regulatory sequence operably linked to a sequence encoding aprotein.

The terms “promoter” and “expression control sequence” are used hereinto refer to an array of nucleic acid control sequences that directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. Promoters include constitutive andinducible promoters. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous expression cassette in a cell is anexpression cassette that is not endogenous to the particular host cell,for example by being linked to nucleotide sequences from an expressionvector rather than chromosomal DNA, being linked to a heterologouspromoter, being linked to a reporter gene, etc.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity of a described target protein, e.g., ligands,agonists, antagonists, and their homologs and mimetics. The term“modulator” includes inhibitors and activators. Inhibitors are agentsthat, e.g., inhibit expression or bind to, partially or totally blockstimulation or protease inhibitor activity, decrease, prevent, delayactivation, inactivate, desensitize, or down regulate the activity ofthe described target protein, e.g., antagonists. Activators are agentsthat, e.g., induce or activate the expression of a described targetprotein or bind to, stimulate, increase, open, activate, facilitate,enhance activation or protease inhibitor activity, sensitize or upregulate the activity of described target protein (or encodingpolynucleotide), e.g., agonists. Modulators include naturally occurringand synthetic ligands, antagonists and agonists (e.g., small chemicalmolecules, antibodies and the like that function as either agonists orantagonists). Such assays for inhibitors and activators include, e.g.,applying putative modulator compounds to cells expressing the describedtarget protein and then determining the functional effects on thedescribed target protein activity, as described above. Samples or assayscomprising described target protein that are treated with a potentialactivator, inhibitor, or modulator are compared to control sampleswithout the inhibitor, activator, or modulator to examine the extent ofeffect. Control samples (untreated with modulators) are assigned arelative activity value of 100%. Inhibition of a described targetprotein is achieved when the activity value relative to the control isabout 80%, optionally 50% or 25, 10%, 5% or 1%. Activation of thedescribed target protein is achieved when the activity value relative tothe control is 110%, optionally 150%, optionally 200, 300%, 400%, 500%,or 1000-3000% or more higher.

The term “allosteric” is used to refer to an effect that affects theactivity of one part of an enzyme (such as an active site) by thebinding of a molecule at a different site (regulatory site) at adifferent location on the enzyme. The binding of non-substrate moleculesat allosteric sites effects the binding kinetics of thesubstrate-binding (active) site. “Allosteric binding sites” arecontained in many enzymes and receptors. As a result of binding toallosteric binding sites, the interaction with the normal ligand may beeither enhanced or reduced. For example, an allosteric binding site in3′-phosphoinositide-dependent kinase-1 (PDK1) is the PDK1 interactingfragment (PIF) binding pocket located between helix C, helix B and the−4 and −5 sheets (Pearl et al., Curr. Opin. Struct. Biol. 12, 761-767(2002); Biondi et al., Biochem. J. 372, 1-13 (2003); Newton et al.,Chem. Rev. 101, 2353-2364 (2001)).

As used herein, “promote” or “increase,” or “promoting” or “increasing”are used interchangeably herein. These terms refer to the increase in ameasured parameter (e.g., activity, expression, glycolysis, glycolyticmetabolism, glucose uptake, biosynthesis downstream of glycolysis) in atreated cell (tissue or subject) in comparison to an untreated cell(tissue or subject). A comparison can also be made of the same cell ortissue or subject between before and after treatment. The increase issufficient to be detectable. In some embodiments, the increase in thetreated cell is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 1-fold, 2-fold, 3-fold, 4-fold or more in comparison to anuntreated cell.

As used herein, “inhibit,” “prevent” or “reduce,” or “inhibiting,”“preventing” or “reducing” are used interchangeably herein. These termsrefer to the decrease in a measured parameter (e.g., activity,expression, mitochondrial respiration, mitochondrial oxidation,oxidative phosphorylation) in a treated cell (tissue or subject) incomparison to an untreated cell (tissue or subject). A comparison canalso be made of the same cell or tissue or subject between before andafter treatment. The decrease is sufficient to be detectable. In someembodiments, the decrease in the treated cell is at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or completely inhibited incomparison to an untreated cell. In some embodiments the measuredparameter is undetectable (i.e., completely inhibited) in the treatedcell in comparison to the untreated cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1E. Generation of human induced pluripotent stem cells fromprimary keratinocytes by single gene, OCT4, and small molecules. (a)Treatment with 0.5 μM PD0325901 (PD) and 0.5 μM A-83-01 (A83)significantly improved generation of iPSCs from primary humankeratinocytes transduced with either 4TFs (4F, OKSM) or 3TFs (3F, OKS).NHEKs were seeded at a density of 100,000 transduced cells per 10 cmdish. (b) Further chemical screens identified PS48, NaB, and theircombination that can substantially enhance reprogramming of primaryhuman keratinocytes transduced with 2TFs (OK). NHEKs were seeded at adensity of 100,000 transduced cells per 10 cm dish. (c) Experimentalscheme for generation of human iPSCs from primary human keratinocytestransduced by a single reprogramming gene, OCT4. KCM, keratinocyteculture medium; hESCM, human ESC culture media. (d) Live immunostainingwith TRA-1-81 of iPSC colonies that were generated from primary humankeratinocytes transduced with 2TFs/OK or 1TF/OCT4 before picking-up ofcolonies. (e) The established human iPSC-OK and iPSC-O cells expresstypical pluripotency markers, including ALP (alkaline phosphatase),OCT4, SOX2, NANOG, SSEA-4 and TRA-1-81. Nuclei were stained with DAPI.

FIG. 2A-2F. In depth characterizations of human iPSC-OK and iPSC-Ocells. (a) Expression analysis by RT-PCR of the endogenous pluripotencygenes and exogenous OCT4 and KLF4. GAPDH was used as an input control.(b) Methylation analysis of the OCT4 and NANOG promoters by bisulfitegenomic sequencing. Open circles and closed circles indicateunmethylated and methylated CpGs in the promoter regions, respectively.(c) Scatter plots comparing global gene expression patterns betweeniPSC-O cells and NHEKs, and hESCs. The positions of the pluripotencygenes OCT4, NANOG, and SOX2 are shown by arrows. Black lines indicatethe linear equivalent and twofold changes in gene expression levelsbetween the samples. (d) Human iPSC-OK and iPSC-O could effectivelydifferentiate in vitro into cells in the three germ layers, includingneural ectodermal cells (βIII tubulin⁺), mesodermal cells (SMA⁺), andendodermal cells (AFP⁺) using EB method. (e) Quantitative PCR test ofthree germ layer markers from differentiated human iPSCs using EBmethod: ectoderm (PAX6, βIII TUBULIN), mesoderm (FOXF1, HAND1) andendoderm (AFP, GATA6). Data denotes GAPDH-normalized fold changesrelative to undifferentiated parental human iPSCs. (f) Human iPSC-OK andiPSC-O could effectively produce full teratoma, which containsdifferentiated cells in the three germ layers, in SCID mice.

FIG. 3A-3F. Generation and characterization of human induced pluripotentstem cells from human umbilical vein endothelial cells by single gene,OCT4, and small molecules. (a) Experimental scheme for generation ofhuman iPSCs from HUVECs transduced by OCT4. HCM, HUVEC culture medium;hESCM, human ESC culture media. (b) The established hiPSC-O cells fromHUVECs express typical pluripotency markers, including NANOG and SSEA-4.Nuclei were stained with DAPI. (c) Expression analysis by RT-PCR of theendogenous pluripotency genes. GAPDH was used as an input control. (d)Methylation analysis of the OCT4 and NANOG promoters by bisulfitegenomic sequencing. Open circles and closed circles indicateunmethylated and methylated CpGs in the promoter regions, respectively.(e) hiPSC-O cells from HUVECs could effectively differentiate in vitrointo cells in the three germ layers, including neural ectodermal cells(βIII tubulin⁺), mesodermal cells (SMA⁺), and endodermal cells (AFP⁺)using EB method. (f) hiPSC-O cells could effectively produce fullteratoma, which contains differentiated cells in the three germ layersin SCID mice.

FIG. 4A-4B. Characterization of human iPSC-O cells from AHEKs. (a) Theestablished hiPSC-O cells from adult keratinocytes express typicalpluripotency markers, including NANOG, SOX2 and SSEA-4. Nuclei werestained with DAPI. (b) These hiPSC-O cells could effectivelydifferentiate in vitro into cells in the three germ layers, includingneural ectodermal cells (βIII tubulin⁺), mesodermal cells (SMA⁺), andendodermal cells (AFP⁺) using EB method.

FIG. 5A-5B. Characterization of hum an iPSC-O cells from AFDCs. (a) Theestablished hiPSC-O cells from amniotic fluid derived cells expresstypical pluripotency markers, including NANOG, SOX2 and SSEA-4. Nucleiwere stained with DAPI. (b) These hiPSC-O cells could effectivelydifferentiate in vitro into cells in the three germ layers, includingneural ectodermal cells (βIII tubulin⁺), mesodermal cells (SMA⁺), andendodermal cells (AFP⁺) using EB method.

FIG. 6. Additional hiPSC cell lines express typical pluripotencymarkers. The other established hiPSC-O cell lines express typicalpluripotency markers, including NANOG and SSEA-4. Nuclei were stainedwith DAPI.

FIG. 7. Feeder-free culture of hiPSC cell lines. hiPSCs were split ontoMatrigel/ECM-coated plates in chemically defined hESC medium aspreviously reported. These hiPSCs could be maintained and expanded in afeeder-free environment. ICC showed the expression of pluripiotencymarkers, OCT4 and SSEA4. Nuclei were stained with DAPI.

FIG. 8A-8B. Genotyping of hiPSCs. RT-PCR analysis using genomic DNAshows that only OCT4 transgene integrated in the genome of hiPSC-O lines(hiPSC-O#1, hiPSC-O#3, hiPSC-O#21, hiPSC-O#26 and hiPSC-O#31). NHEKs (a)and HUVECs (b) were used as negative controls, while vectors were usedas positive controls.

FIG. 9. Integration of the OCT4 transgene in hiPSCs. Genomic DNA (10 μg)were digested with EcoRI and hybridized with the OCT4 cDNA probe (anEcoRI/SpeI fragment of pSin-EF2-OCT4-Pur). Multiple transgenicintegrations were detected.

FIG. 10A-10B. Karyotyping for hiPSC cell lines. Metaphase spread ofhiPSC-O#1 (a) and hiPSC-O#21 (b) show normal karyotype after passage 15.

FIG. 11A-11E. PS48 enhances reprogramming process by facilitating ametabolic switch toward glycolysis. (a) PS48 treatment activated PDK1activity. Western blotting analysis of phosphorylation of Akt (Thr-308)after PS48 (5 μM) or UCN-01(20 nM) treatment. (b) PS48 enhancedreprogramming of NHEKs, while UCN-01 (a PDK1 inhibitor) or2-Deoxy-D-glucose (10 mM) (2-DG, a glycolysis inhibitor) inhibitedreprogramming process. Three factor (Klf, Sox, and Oct)-transduced NHEKswere seeded at a density of 100,000 transduced cells per well, treatedwith compounds for four weeks, and then TRA-1-81 positive colonies werecounted. (c) PS48 treatment facilitated/activated a metabolic switch toglycolysis, while treatment of UCN-01 or 2-DG inhibited glycolysis.NHEKs were treated with either PS48, PS48 and UCN-01, or PS48 and 2-DGfor 8d and then lactate production in the medium was measured as atypical index of glycolysis by using the Lactate Assay Kit (BioVision,Mountain View, Calif., USA). (d) PS48 treatment up-regulated theexpression of several key glycolytic genes, including GLUT1, HK2, PFK1and LDHA. (e) Known small molecules that have been widely used tomodulate mitochondrial respiration, glycolysis metabolism or HIFactivation also showed corresponding consistent effects onreprogramming. Four factor (Klf, Sox, Myc, and Oct)-transduced HUVECswere seeded at a density of 20,000 transduced cells per well, treatedwith metabolism modulating compounds for three weeks and TRA-1-81positive colonies were counted. F2,6P, 10 mM Fructose 2,6-bisphosphate;F6P, 10 mM Fructose 6-phosphate; 6-AN, 10 μM 6-aminonicotinamide; OA, 10μM oxalate; DNP, 1 μM 2,4-dinitrophenol; NOG, 1 μM N-oxalylglycine; QC,1 μM Quercetin; 2-HA, 10 μM 2-Hydroxyglutaric acid; NA, 10 μM nicotinicacid; DMSO was used as a control.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention is based on the surprising discovery that a3′-phosphoinositide-dependent kinase-1 (PDK1) activator greatly improvesefficiency of induction of pluripotency in non-pluripotent mammaliancells. Accordingly, the present invention provides for methods ofinducing pluripotency in non-pluripotent mammalian cells wherein themethod comprises contacting the non-pluripotent cells with a PDK1activator.

The present invention is also based on the surprising discovery thatcompounds that promote glycolytic metabolism as described herein greatlyimprove efficiency of induction of pluripotency in non-pluripotentmammalian cells. It was discovered in the present invention thatcompounds that promote glycolytic metabolism facilitate the metabolicreprogramming from mitochondrial oxidation (mainly used by adult somaticcells) to glycolysis (mainly used by embryonic stem cells (ESCs)),thereby inducing pluripotency in non-pluripotent mammalian cells.Compounds that promote glycolysis or compounds that inhibit or impedemitochondrial respiration/oxidation are therefore useful in inducingpluripotency in non-pluripotent mammalian cells. Further, it wasdiscovered that compounds that promote a process either upstream (e.g.,PDK1 pathway, hypoxia-inducible factor pathway, glucose uptaketransporter pathway) or downstream of glycolysis (e.g., fatty acidssynthesis, lipids synthesis, nucleotides synthesis, and amino acidssynthesis) are useful in inducing pluripotency in non-pluripotentmammalian cells. Accordingly, the present invention provides for methodsof inducing pluripotency in non-pluripotent mammalian cells wherein themethod comprises contacting the non-pluripotent cells with one or morecompounds that promote glycolytic metabolism as described herein.

To date, a large number of different methods and protocols have beenestablished for inducing non-pluripotent mammalian cells into inducedpluripotent stem cells (iPSCs). It is believed that the agents describedherein can be used in combination with essentially any protocol forgenerating iPSCs and thereby improve the efficiency of the protocol.Thus, the present invention provides for incubation of non-pluripotentcells with at least a PDK1 activator, including but not limited to anallosteric PDK1 activator, in combination with any protocol forgenerating iPSCs. In other embodiments, the present invention providesfor incubation of non-pluripotent cells with at least a compound thatpromotes glycolytic metabolism in combination with any protocol forgenerating iPSCs.

As used herein, “efficiency of induction,” with respect to induction ofa non-pluripotent cell into an induced pluripotent stem cell, refers tothe number of non-pluripotent cells that can be converted into iPSCs ina defined time frame, or the amount of time it takes to convert adefined number of non-pluripotent cells into iPSCs, under conditionssufficient for inducing pluripotent stem cells. The improvement inefficiency of an iPSC generation protocol will depend on the protocoland which agents of the invention are used. In some embodiments, theefficiency is improved by at least 10%, 20%, 50%, 75%, 100%, 150%, 200%,300% or more compared to the same protocol without inclusion of theagents of the invention (e.g., a PDK1 activator, e.g., an allostericPDK1 activator, or a compound that promotes glycolytic metabolism, e.g.,PDK1 activators, glycolysis activators, glycolysis substrates,glycolytic intermediates and their metabolic precursors thereof, glucoseuptake transporter activators, mitochondrial respiration modulators suchas oxidative phosphorylation inhibitors, and hypoxia-inducible factoractivators). In some embodiments, efficiency is measured with regard toimprovement of the number of iPSCs generated in a particular time frame(e.g., by comparing the number of iPSCs generated from non-pluripotentcells in a defined time frame under conditions comprising theintroduction of one or more agents of the invention to the number ofiPSCs generated from non-pluripotent cells in the defined time frameunder conditions which do not comprise the introduction of one or moreagents of the invention). In some embodiments, efficiency is measuredwith regard to improvement in the speed by which iPSCs are generated(e.g., by comparing the length of time it takes to generate a definednumber of iPSCs from non-pluripotent cells under conditions comprisingthe introduction of one or more agents of the invention to the length oftime it takes to generate a defined number of iPSCs from non-pluripotentcells under conditions which do not comprise the introduction of one ormore agents of the invention). In some embodiments, efficiency ofinduction is measured under conditions comprising transducingnon-pluripotent cells (e.g., normal human epidermal keratinocytes) withOct4 and Klf4, then culturing the transduced cells in the absence orpresence of one or more agents of the invention (e.g., a PDK1activator), as described in the Examples section below. Induction ofiPSCs from non-pluripotent cells can be measured according to any methodknown in the art, including but not limited to marker analysis (e.g.,using pluripotency markers Tra-1-81 and/or OCT4).

According to the methods of the present invention, specific,context-dependent, treatment regimes can improve reprogrammingefficiency. The effectiveness of a certain treatment regime can depend,in some embodiments, on cell types, cell passage numbers, and exogenoustranscription factors used. For example, in some embodiments, a moresignificant improvement in reprogramming efficiency by a specifictreatment regime can be observed when reprogramming cells transducedwith or in contact with fewer than four exogenous transcription factors,i.e., with one, two, or three exogenous transcription factors, ascompared to reprogramming cells transduced with or in contact with fourexogenous transcription factors.

In general, human cells can take considerably longer (e.g., 6-8 weeks)to be reprogrammed than mouse cells (e.g., about 2 weeks). The effectsof a specific treatment regime, in some embodiments, can be moreexaggerated when reprogramming human cells as compared to mouse cells.Accordingly, when relatively longer periods (e.g., at least 3, 4, 5, 6,or more weeks) are used in reprogramming, a treatment regime, e.g., onethat uses an epigenetic modifier, can be used to improve reprogrammingefficiency.

The inventors have found that epigenetic modifiers can improvereprogramming efficiency. As defined herein, the term “epigeneticmodifier” refers to a methylation modifying agent (i.e., agents thatinduce methylation changes to DNA or histones) and/or an acetylationmodifying agent (i.e., agents that induce acetylation changes to DNA orhistones).

In some embodiments, the methylation modifying agent is a DNAmethylation inhibitor (e.g., a DNA methyltransferase (DNMT) inhibitorsuch as RG108)), histone methylation inhibitor and/or histonedemethylation inhibitor. In some embodiments, the acetylation modifyingagent is a histone deacetylase (HDAC) inhibitor (e.g., valproic acid(VPA), sodium butyrate (NaB), trichostatin A (TSA), or suberoylanilidehydroxamic acid (SAHA)), a histone acetyltransferase (HAT) inhibitor,histone deacetylase and histone acetyltransferase. In some embodiments,epigenetic modifiers are agents that inhibit methyltranferases ordemethylases or agents that activate methyltranferases or demethylases.In some embodiments, the epigenetic modifier is an agent that inhibitshistone H3K9 methylation or promotes H3K9 demethylation, e.g., a G9ahistone methyltransferase such as BIX01294.

Some epigenetic modifiers, however, may also induce celldifferentiation. Accordingly, in some embodiments of the invention,epigenetic modifiers are used only in the earlier stage of thetreatment, e.g., in the first 1, 2, 3, or 4 weeks, in the first half,the first ⅓, the first quarter, or the first ⅕ of the treatment period.By omitting epigenetic modifiers in the later stage of the treatment,e.g., in the last 1, 2, 3, or 4 weeks, in the last half, the last ⅓, thelast quarter, or the last ⅕ of the treatment period, the side effects ofinducing cell differentiation by these epigenetic modifiers can be, atleast partially, avoided.

Alternatively, epigenetic modifiers that do not induce differentiation,or only minimally induce differentiation can be used. For example, whenHDAC inhibitor is used in a treatment regime, a HDAC inhibitor that doesnot induce differentiation, or only minimally induces differentiation,e.g., sodium butyrate, is used.

It is further discovered in the present invention that a treatmentregime using MEK inhibitors can improve reprogramming efficiency. MEKinhibitors also support cell self-renewal of the induced pluripotentcells. Some MEK inhibitors, however, may inhibit cell proliferation.Accordingly, in some embodiments of the invention, MEK inhibitors areused only in the later stage of the treatment, e.g., in the last 1, 2,3, or 4 weeks, in the last half, the last ⅓, the last quarter, or thelast ⅕ of the treatment period. By omitting MEK inhibitors in theearlier stage of the treatment, e.g., in the first 1, 2, 3, or 4 weeks,in the first half, the first ⅓, the first quarter, or the first ⅕ of thetreatment period, cell proliferation is not inhibited in the earlierstage. For example, pluripotency can be induced by contacting anon-pluripotent mammalian cell with a PDK1 activator or with a compoundthat promotes glycolytic metabolism (e.g., a PDK1 activator) in theabsence of a MEK inhibitor in the earlier stage, followed by contactingthe non-pluripotent cell with a PDK1 activator or a compound thatpromotes glycolytic metabolism (e.g., a PDK1 activator) and a MEKinhibitor in the later stage. In some embodiments, the method ofinducing pluripotency comprises contacting the non-pluripotent cell witha PDK1 activator or with a compound that promotes glycolytic metabolism(e.g., a PDK1 activator), a TGFβ receptor/ALK5 inhibitor, and a histonedeacetylase (HDAC) inhibitor in the earlier stage, followed bycontacting the non-pluripotent cell with a PDK1 activator or with acompound that promotes glycolytic metabolism (e.g., a PDK1 activator), aTGFβ receptor/ALK5 inhibitor, a histone deacetylase (HDAC) inhibitor anda MEK inhibitor in the later stage.

II. PDK1 Activators

3′-phosphoinositide-dependent kinase-1 or “PDK1” is a master kinaseassociated with the activation of AKT/PKB and many other AGC kinasesincluding PKC, S6K, SGK. An important role for PDK1 is in the signalingpathways activated by several growth factors and hormones includinginsulin signaling. The structure of PDK1 can be divided into twodomains; the kinase or catalytic domain and the PH domain. The PH domainfunctions mainly in the interaction of PDK1 with phosphatidylinositol(3,4)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate whichis important in localization and activation of some membrane associatedPDK1 substrates including AKT. The kinase domain has three ligandbinding sites; the substrate binding site, the ATP binding site, and thePIF binding pocket. Several PDK1 substrates including S6K and Proteinkinase C require binding at this PIF binding pocket. Small moleculeallosteric activators of PDK1 were shown to selectively inhibitactivation of substrates that require docking site interaction. Thesecompounds do not bind to the active site and allow PDK1 to activateother substrates that do not require docking site interaction. PDK1 isconstitutively active and at present, there are no known inhibitorproteins for PDK1. The activation of PDK1's main effector, AKT, isbelieved to require a proper orientation of the kinase and PH domains ofPDK1 and AKT at the membrane. Phosphoinositide-dependent kinase-1 hasbeen shown to interact with SGK, PRKACA, Sodium-hydrogen exchangeregulatory cofactor 2, PRKCD, Protein kinase Mζ (PKMzeta), PKN2, PRKCI,Protein kinase N1, YWHAH and AKT1.

Exemplary PDK1 activators include sphingosine (King et al., Journal ofBiological Chemistry, 275:18108-18113, 2000). Exemplary allostericactivators of PDK1 include PS48((Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid), PS08((Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid) (Hindie etal., Nature Chemical Biology, 5:758-764, 2009; Huse & Kuriyan, Cell 109:275-282, 2002; Johnson & Lewis, Chem. Rev. 101:2209-2242, 2001), andcompound 1 (2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid)(Engel et al., EMBO J. 25: 5469-5480, 2006); 3,5-diphenylpent-2-enoicacids such as compound 12Z and compound 13Z (12Z:2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid; 13Z:(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (Stroba et al., J. Med.Chem. 52, 4683-4693 (2009)). PS48 has the following formula:

As shown in the Examples, inclusion of a PDK1 activator in cellreprogramming can increase efficiency greatly when used alone andresults in even further efficiency increases when used in combinationwith an HDAC inhibitor. Additional inhibitors, including but not limitedto an ALK5 inhibitor and/or a Mek inhibitor, as shown in the Examples,can also be included in reprogramming, particularly where fewer than thefour transcription factors (Oct4, Klf4, Sox2, and c-Myc) are introducedinto the cell during reprogramming.

III. Compounds that Promote Glycolytic Metabolism

As defined herein, a metabolism modulating compound refers to a compoundthat modulates (e.g., promotes or inhibits) metabolism of carbohydrateor other molecules. Metabolism modulating compounds include compoundsthat promote glycolytic metabolism. As defined herein, a compound thatpromotes glycolytic metabolism refers to a compound that facilitatescellular metabolic reprogramming from mitochondrial oxidation (mainlyused by adult somatic cells) to glycolysis (mainly used by ESCs). Insome embodiments, a compound that promotes glycolytic metabolism is acompound that promotes glycolysis or a compound that promotes a processupstream of glycolysis (e.g., PDK1 pathway, hypoxia-inducible factorpathway, glucose uptake transporter pathway). In some embodiments, acompound that promotes glycolytic metabolism is a compound that inhibitsor impedes mitochondrial respiration. In some embodiments, a compoundthat promotes glycolytic metabolism is a compound that promotes aprocess downstream of glycolysis (e.g., fatty acids synthesis, lipidssynthesis, nucleotides synthesis, and amino acids synthesis). Examplesof compounds that promote glycolytic metabolism include PDK1 activators,glycolysis activators, glycolysis substrates, glycolytic intermediatesand their metabolic precursors thereof, glucose uptake transporteractivators, mitochondrial respiration modulators such as oxidativephosphorylation inhibitors, and hypoxia-inducible factor activators. Insome embodiments, a compound that promotes glycolytic metabolism is nota simple sugar (e.g., a simple sugar commonly used in cell culturemedium). Examples of simple sugars include aldoses such as D-glucose,D-mannose and D-galactose, and ketoses such as D-fructose.

1. Glycolysis Activators

Glycolysis activators (e.g., activators of the glycolytic pathway) areknown in the art. Enzymes associated with glycolysis pathway are knownin the art and include hexokinase, glucokinase, phosphoglucoseisomerase, phosphofructokinase, aldolase, triose phosphate isomerase,glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase,phosphoglyceromutase, enolase, pyruvate kinase and lactatedehydrogenase. In some embodiments, the glycolysis activator (e.g., anactivator of the glycolysis pathway) is an activator of an enzymeassociated with the glycolytic pathway. In some embodiments, aglycolysis activator is an activator of one of three particular enzymesuniquely associated with the glycolytic pathway: hexokinase,phosphofructokinase, and pyruvate kinase.

Examples of hexokinase activators include phosphate, citrate, D-malate,3-phosphoglycerate, catecholamines and catecholamines derivatives. Insome embodiments, the hexokinase activator is an allosteric activator.In some embodiments, the hexokinase activators do not include phosphateor citrate.

Examples of glucokinase activators include GKA1(6-[(3-isobutoxy-5-isopropoxybenzoyl)amino]nicotinic acid; Brocklehurstet al., Diabetes 53:535-541, 2004), GKA2(5-({3-isopropoxy-5-[2-(3-thienyl)ethoxy]benzoyl}amino)-1,3,4-thiadiazole-2-carboxylicacid; Brocklehurst et al., Diabetes 53:535-541, 2004), RO-28-1675(Grimsby et al., Science 301:370-373, 2003), and compound A(N-Thiazol-2-yl-2-amino-4-fluoro-5-(1-methylimidazol-2-yl)thiobenzamide;Kamata et al., Structure 12, 429-438, 2004), LY2121260(2-(S)-cyclohexyl-1-(R)-(4-methanesulfonylphenyl)-cyclopropanecarboxylicacid thiazol-2-ylamide; Efanov et al., Endocrinology, 146:3696-3701,2005). In some embodiments, the glucokinase activator is an allostericactivator. Additional glucokinase activators are disclosed in WO00/058293, WO 01/44216, WO 01/83465, WO 01/83478, WO 01/85706, WO01/85707 and WO 02/08209, WO07/075847, WO07/061923, WO07/053345,WO07/104034, WO07/089512, WO08/079787, WO08/111473, WO09/106203,WO09/140624, WO09/140624, WO08/079787, WO02/046173, WO07/006814,WO07/006760, WO06/058923, WO02/048106, WO07/125103, WO07/125105,WO08/012227, WO08/074694, WO08/078674, WO08/084043, WO08/084044,WO09/127544, WO09/127546, WO07/125103, WO07/125105, WO02/014312,WO04/063179, WO07/006761, WO07/031739, WO08/091770, WO08/116107,WO08/118718, WO09/083553, WO04/052869, WO05/123132, WO04/072066,WO07/117381, WO07/115967, WO08/005964, WO08/154563, WO09/022179,WO09/046784, WO08/005964, WO/10/080333, WO/03/095438, WO/06/016194,WO/05/066145, WO/07/115968, WO/07/143434, WO/08/005914, WO/08/149382,WO/09/018065, WO/09/047798, WO/09/046802, WO/10/029461, WO/08/005914,WO/08/149382, WO/07/143434, WO/10/103438, WO/03/047626, WO/05/095418,WO/08/104994, WO/09/082152, WO/09/082152, WO/05/049019, WO/07/048717,WO/09/042435, and WO/09/042435.

Examples of phosphofructokinase (or fructose-6-P kinase) activatorsinclude fructose 2,6-bisphosphate.

Examples of pyruvate kinase activators include xylulose 5-P, ceramide,an agonist of the A1 adenosine receptors such asN-6-cyclopentyladenosine. Additional pyruvate kinase activators aredisclosed in are disclosed in WO10/042867, WO10/042867, WO99/048490, andWO09/025781.

Examples of phosphoglucoisomerase activators, aldolase activators,glyceraldehyde 3P dehydrogenase activators, triose phosphate isomeraseactivators, phosphoglycerate kinase, enolase, phosphoglycerate mutase,and lactate dehydrogenase are known in the art.

2. Glycolysis Substrates

Examples of glycolysis substrates include glucose 6-phosphate, fructose6-phosphate, fructose 1,6-bisphosphate, glyceraldehyde 3-phosphate,1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, andphosphoenolpyruvate. In some embodiments, a compound that promotesglycolytic metabolism is not a simple sugar (e.g., glucose).

3. Glycolytic Intermediates and the Metabolic Precursors Thereof

Glycolytic intermediates are all variously utilized as for biosynthesisof other important molecules such as fatty acids, lipids, nucleotides,and amino acids. Therefore, as defined herein, compounds that promoteglycolytic metabolism include compounds that promote a process that isdownstream of glycolysis (e.g., fatty acids synthesis, lipids synthesis,nucleotides synthesis, and amino acids synthesis). In some embodiments,compounds that promote glycolytic metabolism include glycolyticintermediates, e.g., glycolytic intermediates that were utilized inthese downstream biosynthesis pathways. In some embodiments, compoundsthat promote glycolytic metabolism include metabolic precursors ofglycolytic intermediates. As defined herein, the term “metabolicprecursors” refers to compounds from which glycolytic intermediates aremetabolically converted, e.g., in a cell, a tissue, or human body.

Examples of glycolytic intermediates include glucose 6-phosphate,fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetonephosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate,3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate,oxaloacetate, pyruvate, and metabolite precursors thereof. In someembodiments, the glycolytic intermediate is nicotinamide adeninedinucleotide (NADH). In some embodiments, the compound that promotesglycolytic metabolism is a metabolic precursor of NADH. In someembodiments, the compound that promotes glycolytic metabolism isnicotinic acid or nicotinamide.

4. Glucose Uptake Transporter Activators

As defined herein, the term “glucose uptake transporter activator”refers to compounds that stimulate or otherwise promote the expressionor activity of a glucose uptake transporter. As defined herein, the term“glucose transporter” refers to proteins that transport compounds(whether glucose, glucose analogs, other sugars such as fructose orinositol, or non-sugars such as ascorbic acids) across a cell membraneand are members of the glucose transporter “family” based on structuralsimilarity (e.g., homology to other glucose transport proteins). Asdefined herein, glucose transporters also include transporter proteinsthat have a primary substrate other than glucose. For example, theglucose transporter GLUTS is primarily a transporter of fructose, and isreported to transport glucose itself with low affinity. Similarly, theprimary substrate for the glucose transporter HMIT is myo-inositol (asugar alcohol). As used herein, the term “glucose transporter,” unlessotherwise specified, includes transporters of fructose and inositol.Examples of glucose uptake transporters include a glucose transporterselected from the groups of GLUT1-12, HMIT and SGLT1-6 transporters.

Examples of glucose uptake transporter activators include insulin,pinitol (see, e.g., WO/2000/071111), 8-bromo-cyclic AMP (see, e.g.,Ogura et al., Journal of Endocrinology, 164:171-178, 2000), arachidonicacid (Fong et al., Cellular Signalling, 8:179-183, 1996), phorbol esterssuch as 12-O-tetra-decanoyl-phorbol 13-acetate (see, e.g., MolecularBrain Research, 15:221-226, 1992).

5. Mitochondrial Respiration Modulators (Oxidative PhosphorylationInhibitors)

As defined herein, the term “mitochondrial respiration” or“mitochondrial oxidation” refers to the oxidation of substrate molecules(e.g., sugars, organic acids, pyruvate, etc.) inside mitochondria. Insome embodiments, a compound that promotes glycolytic metabolism is amitochondrial respiration modulator. A compound that can affect thedegree of mitochondria respiration/oxidation is generally referred toherein as a “mitochondrial respiration modulator” or other similar term.In some embodiments, mitochondrial respiration modulator useful for themethods of the invention is a compound that inhibits or impedesmitochondrial respiration or mitochondrial oxidation. In someembodiments, mitochondrial respiration modulator useful for the methodsof the invention is an oxidative phosphorylation inhibitor.

An oxidative phosphorylation inhibitor of the invention can be anyinhibitor of one or more enzymes of oxidative phosphorylation or anoxidative phosphorylation uncoupler. The oxidative phosphorylationenzymes are known in the art and include enzyme complex I (NADH coenzymeQ reductase), II (succinate-coenzyme Q reductase), III (coenzyme Qcytochrome C reductase), IV (cytochrome oxydase), and V (F0-F1, ATPsynthase).

Inhibitors of enzyme complex I are any known in the art and can include,but are not limited to any of the following: tritylthioalanine,carminomycin, and piperazinedione, rotenone, amytal,1-methyl4-phenylpyridinium (MPP+), paraquat, methylene blue, andferricyanide (the latter 2 are electron acceptors). Inhibitors of enzymecomplex II are any known in the art. Inhibitors of coenzyme Q are anyknown in the art. Inhibitors of enzyme complex III are any known in theart and can include, but are not limited to myxothiazol, antimycin A,ubisemiquinone, cytochrome C, 4,6-diaminotriazine derivatives,metothrexate or electron acceptors such as phenazine methosulfate and2,6-Dichlorophenol-indophenol. Inhibitors of enzyme complex IV are anyknown in the art and can include, but are not limited to cyanide,hydrogen sulfide, azide, formate, phosphine, carbon monoxide and electonacceptor ferricyanide. Inhibitors of enzyme complex V are any known inthe art and can include, but are not limited to 2-hydroxyglutaric acid,VM-26 (4′-demethyl-epipodophyllotoxin thenylidene glucoside),tritylthioalanine, carminomycin, piperazinedione, dinitrophenol,dinitrocresol, 2-hydroxy-3-alkyl-1,4-naphtoquinones, apoptolidinaglycone, oligomycin, ossamycin, cytovaricin, naphtoquinone derivatives(e.g., dichloroallyl-lawsone and lapachol), rhodamine, rhodamine 123,rhodamine 6G, carbonyl cyanide p-trifluoromethoxyphenylhydrazone,rothenone, safranine 0, cyhexatin, DDT, chlordecone, arsenate,pentachlorophenol, benzonitrile, thiadiazole herbicides, salicylate,cationic amphilic drugs (amiodarone, perhexiline), gramicidin,calcimycin, pentachlorobutadienyl-cysteine (PCBD-cys), andtrifluorocarbonylcyanide phenylhydrazone (FCCP). Other inhibitors ofoxidative phorphorylation may include atractyloside, DDT, free fattyacids, lysophospholipids, n-ethylmaleimide, mersanyl, andp-benzoquinone.

Oxidative phosphorylation uncouplers refer to compounds that act asuncouplers of oxidative phosphorylation from electron transport.Examples of oxidative-phosphorylation uncouplers include, but are notlimited to, DNP, 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide(S-13), sodium 2,3,4,5,6-pentachlorophenolate (PCP),4,5,6,7-tetrachloro-2-(trifluoromethyl)-1H-benzimidazole (TTFB),Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid),3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonylcyanide m-chloro phenyl hydrazone (CCCP), Carbonyl cyanidep-[trifluoromethoxy]-phenyl-hydrazone (FCCP), andalpha-(phenylhydrazono)phenylacetonitrile derivatives.phenylacetonitrile derivatives; and weak acids comprising: Weakly AcidicPhenols, benzimidazoles, N-phenylanthranilates, salicylanilides,phenylhydrazones, salicylic acids, acyldi-thiocarbazates, cumarines, andaromatic amines.

6. Hypoxia-Inducible Factor Activators

Activators of hypoxia-inducible factor pathway are known in the art andinclude alkaloids and other amino acid derivatives, inhibitors of HIFasparaginyl hydroxylase (factor-inhibiting HIF, or FIH) and HIF prolylhydroxylases (HPH or PHD), inhibitors of glycogen synthase kinase 3β(GSK3β), nitric oxide (NO) donors, microtubule-depolymerizing agents(MDA), phenolic compounds, terpenes/steroids, and prostaglandin E2(PGE2). Examples of alkaloids and other amino acid derivatives includedeferoxamine and desferri-exochelin DFE 722 SM, Ciclopirox olamine[Loprox®, 6-cyclohexyl-1-hydroxy-4-methyl-2(1H)-pyridone2-aminoethanol], and 8-methyl-pyridoxatin. Examples of inhibitors ofglycogen synthase kinase 3β (GSK3β) include indirubin, derivatives ofindirubin such as 5-iodoindirubin-3′-oxime and5-methylindirubin-3′-oxime. Examples of nitric oxide (NO) donors includeS-nitroso-N-acetyl-D,L-penicillamine (SNAP),3-(hydroxy-1-(1-methylethyl)-2-nitrosohydrazino)-1-propanamine (NOCS),diazen-1-ium-1,2-diolate (NOC-18), S-nitrosoglutathione (GSNO), spermineNONOate (a complex of NO with the natural product spermine),diethylamine NONOate, and diethyltryamine NONOate. Examples ofmicrotubule-depolymerizing agents (MDA) include plant alkaloidsvinblastine, colchicine, and synthetic MDAs such as nocodazole. Examplesof phenolic compounds include dibenzoylmethane (DBM), the flavonoidquercetin (3,3′,4′,5,7-pentahydroxyflavone), (−)-epicatechin-3-gallate(ECG), and (−)-epigallocatechin-3-gallate (EGCG). Examples of terpenesand steroids include sesquiterpene-tropolones (e.g., pycnidione, epoloneA and epolone B), 4-hydroxy estradiol (4-OHE2), dihydrotestosterone,methyltrienolone (R1881), and diterpene ester phorbol 12-O-myristate13-acetate (PMA, also known as 12-O-tetradecanoylphorbol 13-acetate orTPA).

Examples of inhibitors of HIF asparaginyl hydroxylase (factor-inhibitingHIF, or FIH) and HIF prolyl hydroxylases (HPH or PHD) include analoguesof 2-oxoglutarate (20G) such as N-oxalylglycine (5, NOG), esterderivatives of NOG (e.g., DMOG (dimethyl-oxalylglycine)),N-((3-hydroxy-6-chloroquinolin-2-yl)carbonyl)-glycine,3-hydroxypyridine-2-carbonyl-glycine, 3,4-dihydroxybenzoate,pyridine-2,5-dicarboxylate, pyridine-2,4-dicarboxylate,N-oxalyl-2S-alanine, additional analogues of 20G as described in Mole etal., Bioorg Med Chem Lett. 13:2677-80, 2003, alahopcin anddealanylalahopcin, dealanylalahopcin analogues 3-carboxymethyleneN-hydroxy succinimide, 3-carboxy-N-hydroxy pyrollidone, 1-mimosine(L-Mim), ethyl 3,4-dihydroxybenzoate (3,4-DHB), and6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamid (S956711).Additional HIF asparaginyl hydroxylase and HIF prolyl hydroxylasesinhibitors are described in, e.g., Ivan et al., Proc Natl Acad Sci USA99: 13459-13464, 2002, WO03/049686, WO03/080566, and WO06/084210,WO10/056767.

Other activators of HIF pathway include iron chelators (e.g.,deferoxamine, 2,2′-pyridyl, 1,10-phenanthroline, Ca2+ chelator BAPTA(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), transitionmetals (e.g., cobalt, nickel, chromium (VI), and copper), theorganomercurial compound mersalyl, and FG-0041 (a compound that isstructurally related to 1,10-phenanthroline).

Additional activators of HIF pathway include proteins that up-regulateHIF-1 translation. Protein kinase C (PKC) increases the rate of HIF-1αtranscription of and functions in conjunction with thephosphatidylinositol 3-kinase (PI3K) pathway, which also enhances HIF-1αtranslation. The PKC pathway activates expression of the S6 ribosomalprotein, which specifically recognizes mRNA transcripts such as HIF-1α.Via phosphorylation of the S6 protein in normoxic conditions, the ratesof HIF-1α mRNA translation can be greatly increased, effectivelycountering the effects of the proteasome degradation of this subunit andincreasing levels of the HIF-1 complex within the cell. The PI3K pathwayhas been identified as the primary means by which various mediators,such as lipopolysaccharides, affect activation of HIF-1α in vascularsmooth muscle cells and macrophages (Dery et al., Int J Biochem CellBio. 37:535-540, 2004; Page et al., J Biol Chem. 277:48403-48409, 2002).

The macrophage-derived peptide PR39 has been shown to stabilize HIF-1αby decreasing its degradation, resulting in accelerated formation ofvascular structures in vitro and increased myocardial vasculature inmice (Li et al., Nat Med 6: 49-55. 2000). Direct induction of HIF-1 hasbeen achieved by using the N- or C-terminal of ODDD polypeptides thatblock VHL-mediated degradation (Maranchie et al., Cancer Cell 1:247-255, 2002).

HIF pathway activators further include other non-hypoxic physiologicalstimuli such as growth factors, cytokines, and hormones. Examples ofgrowth factors that activate HIF pathway include insulin-like growthfactor (IGF)-1 and IGF-2, IGF-binding protein (IGFBP)-2 and IGFBP-3,EGF, basic fibroblast growth factor (bFGF), and heregulin. Examples ofcytokines that activate HIF pathway include tumor necrosis factor alpha(TNFα), interleukin-1 beta (IL-1β), and IL-1. Examples of hormones thatactivate HIF pathway include the vascular hormones angiotensin II andthrombin, thyroid hormone and follicle-stimulating hormone. Otherphysiological factors such as the redox protein thioredoxin-1 (Trx-1)and oxidized low-density lipoprotein (oxLDL) can also induce HIF-1αprotein and activate HIF-1.

7. PDK1 Activators

In some embodiments, compounds that promote glycolytic metabolism arePDK1 activators. Exemplary PDK1 activators are described herein insection II, supra.

IV. HDAC Inhibitors

Exemplary HDAC inhibitors can include antibodies that bind, dominantnegative variants of, and siRNA and antisense nucleic acids that targetHDAC. HDAC inhibitors include, but are not limited to, TSA (trichostatinA) (see, e.g., Adcock, British Journal of Pharmacology 150:829-831(2007)), VPA (valproic acid) (see, e.g., Munster et al., Journal ofClinical Oncology 25:18S (2007): 1065), sodium butyrate (NaBu) (see,e.g., Han et al., Immunology Letters 108:143-150 (2007)), SAHA(suberoylanilide hydroxamic acid or vorinostat) (see, e.g., Kelly etal., Nature Clinical Practice Oncology 2:150-157 (2005)), sodiumphenylbutyrate (see, e.g., Gore et al., Cancer Research 66:6361-6369(2006)), depsipeptide (FR901228, FK228) (see, e.g., Zhu et al., CurrentMedicinal Chemistry 3(3):187-199 (2003)), trapoxin (TPX) (see, e.g.,Furumai et al., PNAS 98(1):87-92 (2001)), cyclic hydroxamicacid-containing peptide 1 (CHAP1) (see, Furumai supra), MS-275 (see,e.g., Carninci et al., WO2008/126932, incorporated herein byreference)), LBH589 (see, e.g., Goh et al., WO2008/108741 incorporatedherein by reference) and PXD101 (see, Goh, supra). In general at theglobal level, pluripotent cells have more histone acetylation, anddifferentiated cells have less histone acetylation. Histone acetylationis also involved in histone and DNA methylation regulation. In someembodiments, HDAC inhibitors facilitate activation of silencedpluripotency genes.

V. ALK5 Inhibitors

TGFβ receptor (e.g., ALK5) inhibitors can include antibodies to,dominant negative variants of, and antisense nucleic acids that suppressexpression of, TGFβ receptors (e.g., ALK5). Exemplary TGFβ receptor/ALK5inhibitors include, but are not limited to, SB431542 (see, e.g., Inman,et al., Molecular Pharmacology 62(1):65-74 (2002)), A-83-01, also knownas3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide(see, e.g., Tojo et al., Cancer Science 96(11):791-800 (2005), andcommercially available from, e.g., Toicris Bioscience);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO (see, e.g., Dalton et al., WO2008/094597, herein incorporatedby reference), BMP4 (see, Dalton, supra), GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide)(see, e.g., Gellibert et al., Journal of Medicinal Chemistry49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki et al., Cancer Research67(5):2351-2359 (2007)), IN-1130(3-45-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide)(see, e.g., Kim et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville et al., Drug News Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride) (see, e.g., DaCosta et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, e.g., thoselisted in Stiefl et al., WO2008/006583, herein incorporated byreference), SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894;SB-203580; SD-093; Gleevec; 3,5,7,2′,4′-pentahydroxyflavone (Morin);activin-M108A; P144; and soluble TBR2-Fc (see, e.g., Wrzesinski et al.,Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska et al., ActaBiochimica Polonica 52(2):329-337 (2005); and Chang et al., Frontiers inBioscience 12:4393-4401 (2007)). Further, while “an ALK5 inhibitor” isnot intended to encompass non-specific kinase inhibitors, an “ALK5inhibitor” should be understood to encompass inhibitors that inhibitALK4 and/or ALK7 in addition to ALK5, such as, for example, SB-431542(see, e.g., Inman et al., J, Mol. Phamacol. 62(1): 65-74 (2002). Withoutintending to limit the scope of the invention, it is believed that ALK5inhibitors affect the mesenchymal to epithelial conversion/transition(MET) process. TGFβ/activin pathway is a driver for epithelial tomesenchymal transition (EMT). Therefore, inhibiting the TGFβ/activinpathway can facilitate the MET (i.e., reprogramming) process.

In view of the data herein showing the effect of inhibiting ALK5, it isbelieved that inhibition of the TGFβ/activin pathway will have similareffects. Thus, any inhibitor (e.g., upstream or downstream) of theTGFβ/activin pathway can be used in combination with, or instead of,ALK5 inhibitors as described in each paragraph herein. ExemplaryTGFβ/activin pathway inhibitors include but are not limited to: TGFβreceptor inhibitors, inhibitors of SMAD2/3 phosphorylation, inhibitorsof the interaction of SMAD2/3 and SMAD4, and activators/agonists ofSMAD6 and SMAD7. Furthermore, the categorizations described below aremerely for organizational purposes and one of skill in the art wouldknow that compounds can affect one or more points within a pathway, andthus compounds may function in more than one of the defined categories.

Inhibitors of SMAD2/3 phosphorylation can include antibodies to,dominant negative variants of, and antisense nucleic acids that targetSMAD2 or SMAD3. Specific examples of inhibitors include PD169316;SB203580; SB-431542; LY364947; A77-01; and3,5,7,2′,4′-pentahydroxyflavone (Morin). See, e.g., Wrzesinski, supra;Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); andKataoka et al., EP1992360, the contents of each of which is incorporatedherein by reference.

Inhibitors of the interaction of SMAD2/3 and SMAD4 can includeantibodies to, dominant negative variants of, and antisense nucleicacids that target SMAD2, SMAD3 and/or SMAD4. Specific examples ofinhibitors of the interaction of SMAD2/3 and SMAD4 include, but are notlimited to, Trx-SARA, Trx-xFoxHlb and Trx-Lefl. (See, e.g., Cui et al.,Oncogene 24:3864-3874 (2005) and Zhao et al., Molecular Biology of theCell, 17:3819-3831 (2006).)

Activators/agonists of SMAD6 and SMAD7 include, but are not limited to,antibodies to, dominant negative variants of, and antisense nucleicacids that target SMAD 6 or SMAD 7. Specific examples of inhibitorsinclude, but are not limited to, smad7-as PTO-oligonucleotides. See,e.g., Miyazono et al., U.S. Pat. No. 6,534,476, and Steinbrecher et al.,US2005119203, both incorporated herein by reference.

VI. MEK Inhibitors

Inhibitors of MEK can include antibodies to, dominant negative variantsof, and siRNA and antisense nucleic acids that suppress expression of,MEK. Specific examples of MEK inhibitors include, but are not limitedto, PD0325901, (see, e.g., Rinehart, et al., Journal of ClinicalOncology 22: 4456-4462 (2004)), PD98059 (available, e.g., from CellSignaling Technology), U0126 (available, for example, from CellSignaling Technology), SL 327 (available, e.g., from Sigma-Aldrich),ARRY-162 (available, e.g., from Array Biopharma), PD184161 (see, e.g.,Klein et al., Neoplasia 8:1-8 (2006)), PD184352 (CI-1040) (see, e.g.,Mattingly et al., The Journal of Pharmacology and ExperimentalTherapeutics 316:456-465 (2006)), sunitinib (see, e.g., Voss et al.,US2008004287 incorporated herein by reference), sorafenib (see, Vosssupra), Vandetanib (see, Voss supra), pazopanib (see, e.g., Voss supra),Axitinib (see, Voss supra) and PTK787 (see, Voss supra).

Currently, several MEK inhibitors are undergoing clinical trialevaluations. CI-1040 has been evaluated in Phase I and II clinicaltrials for cancer (see, e.g., Rinehart et al., Journal of ClinicalOncology 22(22):4456-4462 (2004)). Other MEK inhibitors being evaluatedin clinical trials include PD184352 (see, e.g., English et al., Trendsin Pharmaceutical Sciences 23(1):40-45 (2002)), BAY 43-9006 (see, e.g.,Chow et al., Cytometry (Communications in Clinical Cytometry) 46:72-78(2001)), PD-325901 (also PD0325901), GSK1120212, ARRY-438162, RDEA119,AZD6244 (also ARRY-142886 or ARRY-886), RO5126766, XL518 and AZD8330(also ARRY-704). See, e.g., information from the National Institutes ofHealth located on the World Wide Web at clinicaltrials.gov as well asinformation from the National Cancer Institute located on the World WideWeb at cancer.gov/clinicaltrials.

VII. Reprogramming

To date, a large number of different methods and protocols have beenestablished for inducing non-pluripotent mammalian cells into inducedpluripotent stem cells (iPSCs). iPSCs are similar to ESCs in morphology,proliferation, and pluripotency, judged by teratoma formation andchimaera contribution. It is believed that PDK1 activators or compoundsthat promote glycolytic metabolism (e.g., PDK1 activators), optionallyin combination with an HDAC inhibitor, and optionally an ALK5 inhibitorand optionally a Mek inhibitor, will improve essentially anyreprogramming protocol for generating iPSCs. Reprogramming protocolsthat can be improved are believed to include those involvingintroduction of one or more reprogramming transcription factors selectedfrom an Oct polypeptide (including but not limited to Oct 3/4), a Soxpolypeptide (including but not limited to Sox2), a Klf polypeptide(including but not limited to Klf4) and/or a Myc polypeptide (includingbut not limited to c-Myc). Thus, in some embodiments, conditionssufficient to induce a cell to become a pluripotent stem cell compriseconditions in which one or more reprogramming transcription factorsselected from an Oct polypeptide (including but not limited to Oct 3/4),a Sox polypeptide (including but not limited to Sox2), a Klf polypeptide(including but not limited to Klf4) and/or a Myc polypeptide (includingbut not limited to c-Myc) are introduced into the cell. As noted in theExamples, PDK1 activators have been shown to improve reprogramming withas few as one reprogramming transcription factor (e.g., Oct4 alone).Thus, in some embodiments, conditions sufficient to induce a cell tobecome a pluripotent stem cell comprise conditions in which onereprogramming transcription factor (e.g., Oct4 alone) is introduced intothe cell.

In some embodiments, conditions sufficient to induce a cell to become apluripotent stem cell comprise introducing reprogramming factors intothe cells, for example, by expression from a recombinant expressioncassette that has been introduced into the target cell, or by incubatingthe cells in the presence of exogenous reprogramming transcriptionfactor polypeptides such that the polypeptides enter the cell.

Studies have shown that retroviral transduction of mouse fibroblastswith four transcription factors that are highly expressed in ESCs(Oct-3/4, Sox2, KLF4 and c-Myc) generate induced pluripotent stem (iPS)cells. See, Takahashi, K. & Yamanaka, S. Cell 126, 663-676 (2006);Okita, K., Ichisaka, T. & Yamanaka, S. Nature 448, 313-317 (2007);Wernig, M. et al. Nature 448, 318-324 (2007); Maherali, N. et al. CellStem Cell 1, 55-70 (2007); Meissner, A., Wernig, M. & Jaenisch, R.Nature Biotechnol. 25, 1177-1181 (2007); Takahashi, K. et al. Cell 131,861-872 (2007); Yu, J. et al. Science 318, 1917-1920 (2007); Nakagawa,M. et al. Nature Biotechnol. 26, 101-106 (2007); Wernig, M., Meissner,A., Cassady, J. P. & Jaenisch, R. Cell Stem Cell. 2, 10-12 (2008).Studies have also demonstrated reprogramming of human somatic cells withtranscription factors that are highly expressed in ESCs: Hockemeyer etal. Cell Stem Cell. 11; 3(3):346-53 (2008); Lowry et al. Proc Natl AcadSci USA. 105(8):2883-8 (2008); Park et al. Nature. 10; 451(7175):141-6(2008); Nakagawa et al. Nat Biotechnol. January; 26(1):101-6 (2008);Takahashi et al. Cell. 131(5):861-72 (2007); and Yu et al. Science.318(5858):1917-20 (2007). Such methods are believed to be improved withthe inclusion of a PDK1 activator or one of more compounds that promoteglycolytic metabolism (e.g., a PDK1 activator) and optionally otheragents as described herein.

To address the safety issues that arise from target cell genomesharboring integrated exogenous sequences, a number of modified geneticprotocols have been further developed and can be used according to thepresent invention. These protocols produce iPS cells with potentiallyreduced risks, and include non-integrating adenoviruses to deliverreprogramming genes (Stadtfeld, M., et al. (2008) Science 322, 945-949),transient transfection of reprogramming plasmids (Okita, K., et al.(2008) Science 322, 949-953), piggyBac transposition systems (Woltjen,K., et al. (2009). Nature 458, 766-770, Yusa et al. (2009) Nat. Methods6:363-369, Kaji, K., et al. (2009) Nature 458, 771-775), Cre-excisableviruses (Soldner, F., et al. (2009) Cell 136, 964-977), andoriP/EBNAl-based episomal expression system (Yu, J., et al. (2009)Science DOI: 10.1126); the contents of each of which is incorporated byreference herein in its entirety. Thus, in some embodiments, conditionssufficient to induce a cell to become a pluripotent stem cell compriseconditions in which reprogramming factors are delivered bynon-integrating adenoviruses, transient transfection of reprogrammingplasmids, piggyBac transposition systems, re-excisable viruses (Soldner,F., et al. (2009) Cell 136, 964-977), and/or oriP/EBNAl-based episomalexpression systems, according to any of the protocols described above.In some embodiments, a PDK1 activator or one of more compounds thatpromote glycolytic metabolism (e.g., a PDK1 activator) and optionallyother agents as described herein are incubated with cells in any of theprotocols described above.

As noted above, reprogramming can involve culturing target cells in thepresence of one or more proteins under conditions to allow forintroduction of the proteins into the cell. See, e.g., Zhou H et al.,Cell Stem Cell. 2009 May 8; 4(5):381-4; WO/2009/117439. One canintroduce an exogenous polypeptide (i.e., a protein provided fromoutside the cell and/or that is not produced by the cell) into the cellby a number of different methods that do not involve introduction of apolynucleotide encoding the polypeptide. In some embodiments, conditionssufficient to induce a cell to become a pluripotent stem cell compriseintroducing into the cell one or more exogenous proteins, each exogenousprotein comprising a transcription factor polypeptide of interest linked(e.g., linked as a fusion protein or otherwise covalently ornon-covalently linked) to a polypeptide that enhances the ability of thetranscription factor to enter the cell (and in some embodiments the cellnucleus).

Examples of polypeptide sequences that enhance transport acrossmembranes include, but are not limited to, the Drosophila homeoproteinantennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34,1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88:1864-8,1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90: 9120-4,1993), the herpes simplex virus structural protein VP22 (Elliott andO'Hare, Cell 88: 223-33, 1997); the HIV-1 transcriptional activator TATprotein (Green and Loewenstein, Cell 55: 1179-1188, 1988; Frankel andPabo, Cell 55: 1 289-1193, 1988); Kaposi FGF signal sequence (kFGF);protein transduction domain-4 (PTD4); Penetratin, M918, Transportan-10;a nuclear localization sequence, a PEP-I peptide; an amphipathic peptide(e.g., an MPG peptide); delivery enhancing transporters such asdescribed in U.S. Pat. No. 6,730,293 (including but not limited to apeptide sequence comprising at least 5-25 or more contiguous arginines(SEQ ID NO:1) or 5-25 or more arginines in a contiguous set of 30, 40,or 50 amino acids; including but not limited to an peptide havingsufficient, e.g., at least 5, guanidino or amidino moieties); andcommercially available Penetratin™ 1 peptide, and the Diatos PeptideVectors (“DPVs”) of the Vectocell® platform available from Daitos S. A.of Paris, France. See also, WO/2005/084158 and WO/2007/123667 andadditional transporters described therein. Not only can these proteinspass through the plasma membrane but the attachment of other proteins,such as the transcription factors described herein, is sufficient tostimulate the cellular uptake of these complexes. A number ofpolypeptides capable of mediating introduction of associated moleculesinto a cell have been described previously and can be adapted to thepresent invention. See, e.g., Langel (2002) Cell Penetrating PeptidesCRC Press, Pharmacology and Toxicology Series.

Exemplary polypeptide sequences that enhance transport across membranesinclude:

VP22: (SEQ ID NO: 2) G S P P T A P T R S K T P A Q G L A R K L H F S TA P P N P D A P W T P R V A G F N K R V F R F S P Q T A R R A T T T R I;kFGF: (SEQ ID NO: 3) A G S G G A A V A L L P A V L L A L L A P G G E FA; PTD4: (SEQ ID NO: 4) A G S G G Y A R A A A R Q A R A G G E F A;PENETRATIN: (SEQ ID NO: 5) R Q I K I W F Q G R R M K W K K TAT:(SEQ ID NO: 6) Y G R K K R R Q R R R; M918: (SEQ ID NO: 7)M V T V L F R R L R I R R A C G P P R V R V; TRANSPORTAN-10:(SEQ ID NO: 8) A G Y L L G K I G L K A L A A L A K K I L.

In some embodiments, the polypeptide that enhances transport acrossmembranes is a peptide sequence comprising at least 5 or more contiguousor non-contiguous arginines (e.g., an 8-arginine peptide; SEQ ID NO:9).In some embodiments, the polypeptide that enhances transport acrossmembranes is a peptide sequence comprising at least 7 or more contiguousor non-contiguous arginines. For example, the polypeptide that enhancestransport across membranes is a peptide sequence comprising 11contiguous arginines (SEQ ID NO:10), e.g., ESGGGGSPGRRRRRRRRRRR (SEQ IDNO:11). As noted above, the arginines in the transport enhancingsequence need not all be contiguous. In some embodiments, thepolyarginine (e.g., the contiguous or non-contiguous) region is at least5, 8, 10, 12, 15, 20, or more amino acids long and has at least e.g.,40%, 50%, 60%, 70%, 80%, 90%, or more arginines.

In some embodiments, conditions sufficient to induce a cell to become apluripotent stem cell comprise conditions in which one or more exogenouspolypeptides, e.g., an Oct polypeptide (including but not limited to Oct3/4), a Sox polypeptide (including but not limited to Sox2), a Klfpolypeptide (including but not limited to Klf4) and/or a Myc polypeptide(including but not limited to c-Myc), is introduced into cells bytraditional methods such as lipofection, electroporation, calciumphosphate precipitation, particle bombardment and/or microinjection, orcan be introduced into cells by a protein delivery agent. For example,the exogenous polypeptide can be introduced into cells by covalently ornoncovalently attached lipids, e.g., by a covalently attached myristoylgroup. Lipids used for lipofection are optionally excluded from cellulardelivery modules in some embodiments. In some embodiments, thetranscription factor polypeptides described herein are exogenouslyintroduced as part of a liposome, or lipid cocktail (such ascommercially available Fugene6 and Lipofectamine). In anotheralternative, the transcription factor proteins can be microinjected orotherwise directly introduced into the target cell. In some embodiments,the transcription factor polypeptides are delivered into cells usingProfect protein delivery reagents, e.g., Profect-P1 and Profect-P2(Targeting Systems, El Cajon, Calif.), or using Pro-Ject® transfectionreagents (Pierce, Rockford Ill., USA). In some embodiments, thetranscription factor polypeptides are delivered into cells using asingle-wall nano tube (SWNT).

As discussed in the Examples of WO/2009/117439, incubation of cells withthe transcription factor polypeptides of the invention for extendedperiods can be toxic to the cells. Therefore, in some embodiments of theinvention, conditions sufficient to induce the non-pluripotent mammaliancell to become a pluripotent stem cell comprise incubating a PDK1activator or one of more compounds that promote glycolytic metabolism(e.g., a PDK1 activator) and optionally an HDAC inhibitor, an ALK5inhibitor and/or a Mek inhibitor, and intermittently incubating thenon-pluripotent mammalian cell with one or more of an Oct polypeptide(including but not limited to Oct 3/4), a Sox polypeptide (including butnot limited to Sox2), a Klf polypeptide (including but not limited toKlf4) and/or a Myc polypeptide (including but not limited to c-Myc) withintervening periods of incubation of the cell in the absence of the oneor more polypeptides. In some embodiments, the cycle of incubation withand without the polypeptides can be repeated for 2, 3, 4, 5, 6, or moretimes and is performed for sufficient lengths of time (i.e., theincubations with and without proteins) to achieve the development ofpluripotent cells.

The various agents (e.g., PDK1 activator or compounds that promoteglycolytic metabolism, HDAC inhibitor, TGFβ receptor/ALK5 inhibitor,MEK/ERK pathway inhibitor, and/or Rho GTPase/ROCK inhibitor, etc.) canbe contacted to non-pluripotent cells either prior to, simultaneouslywith, or after delivery of, programming transcription factors (forexample, delivered via expression cassette or as proteins). Forconvenience, the day the reprogramming factors are delivered isdesignated “day 1.” In some embodiments, the inhibitors are contacted tocells in aggregate (i.e., as a “cocktail”) at about days 3-7 andcontinued for 7-14 days. Alternatively, in some embodiments, thecocktail is contacted to the cells at day 0 (i.e., a day before thepreprogramming factors) and incubated for about 14-30 days.

The cell into which a protein of interest is introduced can be amammalian cell. The cells can be human or non-human (e.g., primate, rat,mouse, rabbit, bovine, dog, cat, pig, etc.). The cell can be, e.g., inculture or in a tissue, fluid, etc. and/or from or in an organism. Cellsthat can be induced to pluripotency include, but are not limited to,keratinocyte cells, hair follicle cells, HUVEC (Human Umbilical VeinEndothelial Cells), cord blood cells, neural progenitor cells andfibroblasts.

In some embodiments, small molecules can improve the efficiency of aprocess for generating pluripotent cells (e.g., iPS cells). For example,improved efficiency can be manifested by speeding the time to generatesuch pluripotent cells (e.g., by shortening the time to development ofpluripotent cells by at least a day compared to a similar or sameprocess without the small molecule). Alternatively, or in combination, asmall molecule can increase the number of pluripotent cells generated bya particular process (e.g., increasing the number in a given time periodby at least 10%, 30%, 50%, 100%, 200%, 500%, etc. compared to a similaror same process without the small molecule).

Optionally, or in addition, small molecules can “complement” or replacewhat is generally otherwise understood as a necessary expression of oneof these proteins to result in pluripotent cells. By contacting a cellwith an agent that functionally replaces one of the transcriptionfactors, it is possible to generate pluripotent cells with all of theabove-listed transcription factors except for the transcription factorreplaced or complemented by the agent.

VIII. Transformation

This invention employs routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)). In some embodiments, expression cassettesfor expression of one or more reprogramming transcription factor isintroduced into a cell.

In some embodiments, the species of cell and protein to be expressed isthe same. For example, if a mouse cell is used, a mouse ortholog isintroduced into the cell. If a human cell is used, a human ortholog isintroduced into the cell.

It will be appreciated that where two or more proteins are to beexpressed in a cell, one or multiple expression cassettes can be used.For example, where one expression cassette expresses multiplepolypeptides, a polycistronic expression cassette can be used.

Any type of vector can be used to introduce an expression cassette ofthe invention into a cell. Exemplary vectors include but are not limitedto plasmids and viral vectors. Exemplary viral vectors include, e.g.,adenoviral vectors, AAV vectors, and retroviral (e.g., lentiviral)vectors.

Suitable methods for nucleic acid delivery for transformation of a cell,a tissue or an organism for use with the current invention are believedto include virtually any method by which a nucleic acid (e.g., DNA) canbe introduced into a cell, a tissue or an organism, as described hereinor as would be known to one of ordinary skill in the art (e.g.,Stadtfeld and Hochedlinger, Nature Methods 6(5):329-330 (2009); Yusa etal., Nat. Methods 6:363-369 (2009); Woltjen, et al., Nature 458, 766-770(9 Apr. 2009)). Such methods include, but are not limited to, directdelivery of DNA such as by ex vivo transfection (Wilson et al., Science,244:1344-1346, 1989, Nabel and Baltimore, Nature 326:711-713, 1987),optionally with Fugene6 (Roche) or Lipofectamine (Invitrogen), byinjection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448,5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, eachincorporated herein by reference), including microinjection (Harland andWeintraub, J. Cell Biol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215,incorporated herein by reference); by electroporation (U.S. Pat. No.5,384,253, incorporated herein by reference; Tur-Kaspa et al., Mol. CellBiol., 6:716-718, 1986; Potter et al., Proc. Nat'l Acad. Sci. USA,81:7161-7165, 1984); by calcium phosphate precipitation (Graham and VanDer Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol.,7(8):2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990);by using DEAE-dextran followed by polyethylene glycol (Gopal, Mol. CellBiol., 5:1188-1190, 1985); by direct sonic loading (Fechheimer et al.,Proc. Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by liposome mediatedtransfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190,1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979;Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene,10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al.,J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection(Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987); and any combination of such methods, each of whichis incorporated herein by reference.

IX. Mixtures

As discussed herein, the present invention provides for mammalian cellsin a mixture with a PDK1 activator or a compound that promotesglycolytic metabolism, and one or more of (a) a TGFβ receptor/ALK5inhibitor; (b) a MEK inhibitor; (c) a histone deacetylase (HDAC)inhibitor; or (d) an exogenous polypeptide selected from an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide. In some embodiments, the compound that promotes glycolyticmetabolism is a PDK1 activator. In some embodiments, the PDK1 activatoris an allosteric PDK1 activator, e.g., PS48. In some embodiments, thecompound that promotes glycolytic metabolism is a glycolysis activator,e.g., fructose 2,6-bisphosphate. In some embodiments, the compound thatpromotes glycolytic metabolism is a substrate for glycolysis, e.g.,fructose 6-phosphate. In some embodiments, the compound that promotesglycolytic metabolism is a glycolytic intermediate or its metabolicprecursors, e.g., nicotinic acid, NADH, or fructose 6-phosphate. In someembodiments, the compound that promotes glycolytic metabolism is aglucose uptake transporter activator. In some embodiments, the compoundthat promotes glycolytic metabolism is a mitochondrial respirationmodulator. In some embodiments, the mitochondrial respiration modulatoris an oxidative phosphorylation inhibitor, e.g., 2,4-dinitrophenol, or2-hydroxyglutaric acid. In some embodiments, the compound that promotesglycolytic metabolism is a hypoxia-inducible factor activator, e.g.,N-oxalylglycine, or quercetin.

In some embodiments, the mixture further comprises a TGFβ receptor/ALK5inhibitor. TGFβ receptor/ALK5 inhibitors include but are not limited toA-83-01. In some embodiments, the mixture further comprises a MEKinhibitor. MEK inhibitors include but are not limited to PD0325901. Insome embodiments, the mixture further comprises a histone deacetylase(HDAC) inhibitor. HDAC inhibitors include but are not limited to sodiumbutyrate (NaB) and valproic acid (VPA). In some embodiments, the mixturefurther comprises an exogenous transcription factor, e.g., an exogenoustranscription factor selected from an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor comprises an amino acidsequence that enhances transport across cell membranes.

In some embodiments, the compound (e.g., the PDK1 activator or thecompound that promotes glycolytic metabolism) is present in the mixtureat a concentration sufficient to induce or improve efficiency ofinduction to pluripotency. For example, in some embodiments, thecompounds are in a concentration of at least 0.1 nM, e.g., at least 1,10, 100, 1000, 10,000, or 100,000 nM, e.g., between 0.1 nM and 100,000nM, e.g., between 1 nM and 10,000 nM, e.g., between 10 nM and 10,000 nM.In some embodiments, the mixtures are in a synthetic vessel (e.g., atest tube, Petri dish, etc.). Thus, in some embodiments, the cells areisolated cells (not part of an animal). In some embodiments, the cellsare isolated from an animal (human or non-human), placed into a vessel,contacted with one or more compound as described herein. The cells canbe subsequently cultured and optionally, inserted back into the same ora different animal, optionally after the cells have been stimulated tobecome a particular cell type or lineage. In some embodiments, theconcentration of the inhibitors is sufficient to improve by at least10%, 20%, 50%, 75%, 100%, 150%, 200%, 300% or more, the efficiency ofinduction of non-pluripotent cells in the mixture into inducedpluripotent stem cells when the mixture is submitted to conditionssufficient to induce conversion of the cells into induced pluripotentstem cells.

As explained herein, in some embodiments, the cells comprise anexpression cassette for heterologous expression of at least one or moreof an Oct polypeptide, a Myc polypeptide, a Sox polypeptide and a Klfpolypeptide. In some embodiments, the cells do not include an expressioncassette to express one or more (including in some embodiments, any) ofthe Oct, Myc, Sox, or Klf polypeptides.

The cells according to the present invention can be human or non-human(e.g., primate, rat, mouse, rabbit, bovine, dog, cat, pig, etc.).Examples of non-pluripotent cells include those described herein,including but not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, T-cells,keratinocyte cells, hair follicle cells, human umbilical veinendothelial cells (HUVEC), cord blood cells, and neural progenitorcells. In some embodiments, at least 99% of the cells in the mixture areinitially non-pluripotent cells. In some embodiments, essentially all ofthe cells in the mixture are initially non-pluripotent cells.

In some embodiments, at least 0.001%, at least 0.002%, at least 0.005%,at least 0.01%, at least 0.05%, at least 0.1%, at least 0.5%, least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or at least 90% of the cells in the mixture areinduced into pluripotent cells. In some embodiments, at least 99% of thecells in the mixture are induced into pluripotent cells. In someembodiments, essentially all of the cells are induced intonon-pluripotent cells.

X. Kits

The present invention provides a kit for inducing pluripotency in anon-pluripotent mammalian cell comprising a PDK1 activator or a compoundthat promotes glycolytic metabolism, and one or more of (a) a TGFβreceptor/ALK5 inhibitor; (b) a MEK inhibitor; (c) a histone deacetylase(HDAC) inhibitor; or (d) an exogenous polypeptide selected from an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide. In some embodiments, the compound that promotes glycolyticmetabolism is a PDK1 activator. In some embodiments, the PDK1 activatoris an allosteric PDK1 activator, e.g., PS48. In some embodiments, thecompound that promotes glycolytic metabolism is a glycolysis activator,e.g., fructose 2,6-bisphosphate. In some embodiments, the compound thatpromotes glycolytic metabolism is a substrate for glycolysis, e.g.,fructose 6-phosphate. In some embodiments, the compound that promotesglycolytic metabolism is a glycolytic intermediate or its metabolicprecursors, e.g., nicotinic acid, NADH, or fructose 6-phosphate. In someembodiments, the compound that promotes glycolytic metabolism is aglucose uptake transporter activator. In some embodiments, the compoundthat promotes glycolytic metabolism is a mitochondrial respirationmodulator. In some embodiments, the mitochondrial respiration modulatoris an oxidative phosphorylation inhibitor, e.g., 2,4-dinitrophenol, or2-hydroxyglutaric acid. In some embodiments, the compound that promotesglycolytic metabolism is a hypoxia-inducible factor activator, e.g.,N-oxalylglycine, or quercetin.

In some embodiments, the kit further comprises a TGFβ receptor/ALK5inhibitor, e.g., A-83-01. In some embodiments, the kit further comprisesa MEK inhibitor, e.g., PD0325901. In some embodiments, the kit furthercomprises a histone deacetylase (HDAC) inhibitor, e.g., sodium butyrate(NaB), or valproic acid (VPA). In some embodiments, the kit furthercomprises an exogenous transcription factor, e.g., an exogenoustranscription factor selected from an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide. In someembodiments, the exogenous transcription factor comprises an amino acidsequence that enhances transport across cell membranes.

In some embodiments, the kits further comprise non-pluripotent cells.Examples of non-pluripotent cells include those described herein,including but not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, T-cells,keratinocyte cells, hair follicle cells, human umbilical veinendothelial cells (HUVEC), cord blood cells, and neural progenitorcells.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1: Reprogramming of Human Primary Somatic Cells by OCT4 andChemical Compounds

Here we report a novel small molecule cocktail that enablesreprogramming of human primary somatic cells to iPSCs with exogenousexpression of only OCT4.

Results

Among several readily available primary human somatic cell types,keratinocytes that can be easily isolated from human skin or hairfollicle represent an attractive cell source for reprogramming, becausethey endogenously express KLF4 and cMYC, and were reported to bereprogrammed more efficiently using the conventional four TFs or threeTFs (without MYC) (Aasen, T. et al., Nat Biotechnol 26:1276-1284 (2008);Maherali, N. et al., Cell Stem Cell 3, 340-345(2008)). More recently, wereported that dual inhibition of TGFβ and MAPK/ERK pathways using smallmolecules (i.e., SB431542 and PD0325901, respectively) provides adrastically enhanced condition for reprogramming of human fibroblastswith four exogenous TFs (i.e., Oct4, Sox2, Klf4, and c-Myc TFs, or“OSKM”) (Lin, T. et al., Nat Methods 6:805-808 (2009)). Furthermore, wehave shown that such dual pathway inhibition could also enhancereprogramming of human keratinocytes by two exogenous TFs (i.e., Oct4and Klf4, or “OK”) with two small molecules, Parnate (an inhibitor oflysine-specific demethylase 1) and CHIR99021 (a GSK3 inhibitor) (Li, W.et al., Stem Cells 27:2992-3000 (2009)). However, such a 2-TFsreprogramming process was very inefficient and complex (e.g., involvingtwo exogenous TFs and four chemicals), and reprogramming with even oneless TF appeared daunting. Toward the OCT4 only reprogramming, wedeveloped a step-wise strategy in refining reprogramming condition andidentifying new reprogramming chemical entities.

We first attempted to further optimize the reprogramming process underthe four or three TFs (i.e., OSKM or OSK) condition in neonatal humanepidermal keratinocytes (NHEKs) by testing various inhibitors of TGFβand MAPK pathways at different concentrations using previously reportedhuman iPSC characterization methods (Lin, T. et al., Nat Methods6:805-808 (2009)). We found that the combination of 0.5 μM PD0325901 and0.5 μM A-83-01 (a more potent and selective TGFβ receptor inhibitor) wasmore effective in enhancing reprogramming of human keratinocytestransduced with OSKM or OSK (FIG. 1a ). Remarkably, when we furtherreduced viral transductions to only two factors/OK, we could stillgenerate iPSCs from NHEKs when they were treated with 0.5 μM PD0325901and 0.5 μM A-83-01, although with low efficiency. Then we beganscreening additional small molecules from a collection of knownbioactive compounds at various concentrations as previously reported.Among dozens of compounds tested so far, surprisingly we found that asmall molecule activator of PDK1 (3′-phosphoinositide-dependentkinase-1), PS48 (5 μM) that has never been reported in reprogramming,can significantly enhance the reprogramming efficiency about fifteenfold. Interestingly, we also found that 0.25 mM sodium butyrate (NaB, ahistone deacetylase inhibitor) turned out to be much more reliable andefficient than the previously reported 0.5 mM VPA for the generation ofiPSCs under OK condition (FIG. 1b ). Subsequent follow-up studiesdemonstrated that the combination of 5 μM PS48 and 0.25 mM NaB couldfurther enhance the reprogramming efficiency over twenty-five fold (FIG.1b and Table 3).

With such unprecedented efficiency in reprogramming NHEKs under only twoTFs, we further explored the possibility of generating iPSCs with OCT4alone by refining combinations of those small molecules during differenttreatment windows. Primary NHEKs were transduced with OCT4 and treatedwith chemicals (FIG. 1c ). Among various conditions, small iPSC coloniesresembling hESCs (four to six colonies out of 1,000,000 seeded cells)appeared in OCT4 infected NHEKs that were treated with 0.25 mM NaB, 5 μMPS48 and 0.5 μM A-83-01 during the first four weeks, followed bytreatment with 0.25 mM NaB, 5 μM PS48, 0.5 μM A-83-01 and 0.5 μMPD0325901 for another four weeks (FIG. 1c ). Such TRA-1-81 positive iPSCcolonies (FIG. 1d ) grew larger under conventional hESC culture mediaand could be serially passaged to yield stable iPSC clones that werefurther characterized (FIGS. 1e and 2). In addition, OCT4 only iPSCscould also be generated from human adult keratinocytes by addition of 2μM Parnate and 3 μM CHIR99021 (which had been shown to improvereprogramming of NHEKs under OK condition) to this chemical cocktail.After the reliable reprogramming of primary keratinocytes to iPSCs byOCT4 and small molecules, we further applied the conditions to otherhuman primary cell types, including HUVECs (differentiated mesodermcells) and AFDCs (amniotic fluid derived cells). Similarly, TRA-1-81positive iPSC colonies appeared in OCT4 infected HUVECs and AFDCs thatwere treated with chemicals. Remarkably, it appeared that reprogrammingof HUVECs and AFDCs was more efficient and faster than reprogramming ofNHEKs under the OCT4 and small molecule conditions (Table 3). Two clonesof iPSCs from each cell type were long-term expanded for over 20passages under conventional hESC culture condition and furthercharacterized (Table 4).

These stably expanded hiPSC-OK and hiPSC-O cells are morphologicallyindistinguishable from hESCs, and could be cultured on ECM-coatedsurface under feeder-free and chemically defined conditions (FIG. 1e andFIG. 6). They stained positive for alkaline phosphatase (ALP) andexpressed typical pluripotency markers, including OCT4, SOX2, NANOG,TRA-1-81 and SSEA4, detected by immunocytochemistry/ICC (FIG. 1e, 3b ,FIGS. 4-5). In addition, RT-PCR analysis confirmed the expression of theendogenous human OCT4, SOX2, NANOG, REX1, UTF1, TDGF2, FGF4 genes, andsilencing of exogenous OCT4 and KLF4 (FIGS. 2a and 3c ). Furthermore,bisulfite sequencing analysis revealed that the OCT4 and NANOG promotersof hiPSC-OK and hiPSC-O cells are largely demethylated (FIGS. 2b and 3d). This result provides further evidence for reactivation of thepluripotency transcription program in the hiPSC-OK and hiPSC-O cells.Global gene expression analysis of hiPSC-O cells, NHEKs and hESCs showedthat hiPSC-O cells are distinct from NHEKs (Pearson correlation value:0.87) and most similar to hESCs (Pearson correlation value: 0.98) (FIG.2c ). Genotyping analysis showed that hiPSC-O cells only contained theOCT4 transgene without the contamination of transgene KLF4 or SOX2 (FIG.8). Southern blot analysis showed that there were multiple differentintegration sites of the OCT4 transgene (FIG. 9) among different clones.In addition, karyotyping result demonstrated that hiPSC-O maintainednormal karyotype during the whole reprogramming and expansion process(FIG. 10). Furthermore, DNA fingerprinting test excluded the possibilitythat these hiPSCs arose from hESC contamination in the laboratory (Table5).

To examine the developmental potential of these hiPSC-O cells, they weredifferentiated in vitro by the standard embryoid body (EB)differentiation method. ICC analyses demonstrated that they couldeffectively differentiate into βIII-tubulin⁺ characteristic neuronalcells (ectoderm), SMA⁺ mesodermal cells, and AFP⁺ endodermal cells(FIGS. 2d and 3e ). Quantitative PCR analyses further confirmed theexpression of these and additional lineage specific marker genes,including ectodermal cells (βIII-tubulin and NESTIN), mesodermal cells(MSX1 and MLC2a), and endodermal cells (FOXA2 and AFP) (FIG. 2e ).Following EB protocol, these hiPSC-OK and hiPSC-O cells could also giverise to rhythmically beating cardiomyocytes. To test their in vivopluripotency, they were transplanted into SCID mice. Four to six weekslater, these hiPSC-O cells effectively generated typical teratomascontaining derivatives of all three germ layers (FIGS. 2f and 3f ).Collectively, these in vitro and in vivo characterizations demonstratedthat a single transcription factor, OCT4, combined with a defined smallmolecule cocktail is sufficient to reprogram several human primarysomatic cells to iPSCs that are morphologically, molecularly andfunctionally similar to pluripotent hESCs.

Discussion

The studies presented above have a number of important implications:First, although fetal NSCs were shown to be reprogrammed to iPSCs byectopic expression of OCT4 alone, there has been significant skepticismabout whether exogenous OCT4 gene alone would be sufficient to reprogramother more practical human somatic cells that do not endogenouslyexpress SOX2 (one of the two master pluripotency genes inreprogramming), are at later developmental stages (e.g., earlyembryonic/fetal vs. born/adult), and can be obtained without significantharms to the individual. To our knowledge, our study is the firstdemonstration that iPSCs can be practically derived from readilyavailable primary human somatic cells (e.g., keratinocytes) transducedwith a single exogenous reprogramming gene, OCT4. In contrast to neuralstem cells from the brain, keratinocytes are more accessible and can beeasily obtained from born individuals with less invasive procedures.This further strengthens the strategy of exploiting various practicallyaccessible human somatic cells for iPSC generation with safer approachesand/or better qualities. Thus, this new method and its furtherdevelopment would significantly facilitate production ofpatient-specific pluripotent stem cells for various applications.

Second, although small molecules and their combinations have beenidentified to replace only one or two reprogramming TFs, it becomesexponentially challenging to generate iPSCs when more exogenousreprogramming TFs are omitted together. The identification of this newsmall molecule cocktail, which functionally replaces three mastertranscription factors all together (i.e., SOX2, KLF4 and MYC) inenabling generation of iPSCs with OCT4 alone, represents another majorstep toward the ultimate reprogramming with only small molecules, andfurther proved and solidified the chemical approach to iPSCs.

Third, this demonstrated single gene condition also has a significantimplication for protein-induced pluripotent stem cell (piPSC)technology. A practical challenge for piPSC technology is large-scaleand reliable production of the four transducible reprogramming proteins,each of which behaves differently in manufacture (e.g., theirexpression, folding, stability etc.). Clearly, combining this smallmolecule cocktail with a single transducible protein would significantlysimplify the piPSC technology and facilitate its applications.

Fourth, we identified a new small molecule, PS48, with a newtarget/mechanism in enhancing reprogramming. PS48 is an allosteric smallmolecule activator of PDK1 (Hindie, V. et al., Nat Chem Biol 4:758-764(2009)). One mechanism by which PS48 enhances reprogramming appears tobe facilitating the metabolic reprogramming from mitochondrial oxidationmainly used by adult somatic cells to glycolysis mainly used by ESCs(which is also known as the Warburg effect) (Manning, B. D. and Cantley,Cell 129:1261-1274 (2007); Kondoh, H. et al., Antioxid Redox Signal9:293-299 (2007); Heiden, M. G. V. et al., Science 324:1029-1033(2009)). Such differential use of glycolytic metabolism overmitochondrial respiration by pluripotent stem cells would favorpluripotency by promoting proliferation/cell cycle transition with lessoxidative stress. For highly proliferating cells, oxidativephosphorylation would not be able to meet the demand of providingmacromolecular precursors for cell replication, but also generatessignificant amount of reactive oxygen species in mitochondria that couldinduce excessive oxidative damages. On the other hand, glycolyticmetabolism could more effectively generate macromolecular precursors,such as glycolytic intermediates for nonessential amino acids andacetyl-CoA for fatty acids, while provide sufficient energies to meetthe needs of proliferating cells (Kondoh, H. et al., Antioxid RedoxSignal 9:293-299 (2007); Heiden, M. G. V. et al., Science 324:1029-1033(2009)). Interestingly, hypoxic condition and its effector HIF-1αactivation not only have been closely linked to promoting glycolyticmetabolism, but also were shown to enhance both mouse and humanreprogramming (Yoshida, Y. et al., Cell Stem Cell 5:237-241 (2009)).Mechanistically, growth factor signaling pathways, hypoxiccondition/HIF-1α and reprogramming factor Myc appear to regulatecomplementary aspects of cellular metabolism, including up-regulatingglucose transporters and metabolic enzymes of glycolysis, such as GLUT1,HK2 and PFK1 (Gordan, J. D. et al., Cancer Cell 12:108-113 (2007);DeBerardinis, R. J. et al., Cell Metabolism 7:11-20 (2008)). Thosestudies suggest that one potential conserved mechanism of Myc, hypoxiccondition/HIF-1α, and growth factors/Akt pathway activation in enhancingreprogramming converge on their essential roles in regulating glycolyticmetalolism. Supporting this notion, we found that treatment with PS48activated down-stream Akt/PKB (FIG. 11a ), and up-regulated expressionof several key glycolytic genes (FIG. 11d ), facilitating the metabolicswitch to glycolysis (FIG. 11c ). Conversely, we found that inactivationof PDK1 activity by UCN-01 (a PDK1 inhibitor) or inhibition ofglycolysis by 2-Deoxy-D-glucose (a glycolysis inhibitor) not onlyattenuated glycolysis (FIG. 11c ) but also blocked reprogramming process(FIG. 11b ). Furthermore, several known small molecules that have beenwidely used to modulate mitochondrial respiration (2,4-dinitrophenol),glycolytic metabolism (Fructose 2,6-bisphosphate and oxalate), or morespecifically HIF pathway activation (N-oxalylglycine and Quercetin) alsoshowed corresponding consistent effects on reprogramming: i.e.,compounds facilitating glycolytic metabolism enhance reprogramming (suchas 2,4-dinitrophenol and N-oxalylglycine), while compounds blockingglycolytic metablism inhibit reprogramming (such as oxalate) (FIG. 11e )(Hewitson, K. S. and Schofield, C. J., Drug Discov Today 9:704-711(2004); Pelicano, H. et al., Oncogene 25:4633-4646 (2006)). Inconclusion, these results indicated that a metabolic switch to anaerobicglycolysis is critical for and facilitate reprogramming of somatic cellsto pluripotent stem cells.

Finally, this new and powerful small molecule cocktail for reprogrammingvalidated the step-wise chemical optimization and screening strategypresented here as a productive approach toward the ultimate purelychemical-induced pluripotent stem cells. Moreover, we found thatdifferent small molecules modulating the same target/mechanism couldhave significantly different effects on reprogramming in a differentcontext, exemplified by A-83-01's and NaB's better reprogrammingenhancing activities in human keratinocytes, suggests the importance of“individualized” optimization and treatment with different regimens forspecific reprogramming context.

Methods

Cell Culture

Normal Human Epidermal Keratinocytes (Lonza) were maintained inKeratinocyte culturing medium (KCM, Lonza). Human Umbilical VeinEndothelial Cells (HUVECs, Millipore) were maintained in EndoGRO-VEGFComplete Medium (HCM, CHEMICON). Human ESCs and hiPSCs were cultured onMEF feeder cells in conventional human ESC culture media (hESCM:DMEM/F12, 15% Knockout serum replacement, 1% Glutamax, 1% Non-essentialamino acids, 1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol and 10ng/ml bFGF). All cell culture products were from Invitrogen/Gibco BRLexcept where mentioned.

Lentivirus Production

The lentivirus supernatants were produced and harvested as previouslydescribed (Yu, J. et al., Science 318:1917-1920 (2007)). The plasmidsused for lentivirus production include pSin-EF2-Puro-hOCT4,pSin2-EF2-Puro-hSOX2, pLove-mKlf4, pLove-mMyc, the packaging plasmidpsPAX2 and the envelop-coding plasmid pMD2.G (Yu, J. et al., Science318:1917-1920 (2007) and Li, W. et al., Stem Cells 27:2992-3000 (2009)).

Reprogramming of NHEKs

NHEKs were cultured in a 100 mm tissue culture dish and transduced 3times (3-4 hours each transduction) with freshly produced lentivirussupernatants. 1,000,000 transduced NHEKs were seeded on the irradiatedx-ray inactivated CF1 MEF feeder cells in a 100-mm dish and cultured inKCM and treated with 5 μM PS48, 0.25 mM NaB (Stemgent) and 0.5 μMA-83-01 (Stemgent) for 2 weeks, followed by changing half volume ofmedia to hESCM and supplementing with 5 μM PS48, 0.25 mM NaB and 0.5 μMA-83-01 for another 2 weeks. Then cell culture media were changed tohESCM and supplemented with 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01 and0.5 μM PD0325901 (Stemgent) for an additional four weeks. The same OCT4infected keratinocytes cultured in media without chemicals were used asa control. The culture was split by Accutase (Millipore) and treatedwith 1 μM Thiazovivin (Stemgent) in the first day after splitting. TheiPSC colonies stained positive by Alexa Fluor 555 Mouse anti-HumanTRA-1-81 antibody (BD Pharmingen) were picked up for expansion on feedercells in hESCM and cultured routinely.

Reprogramming of HUVECs

HUVECs were cultured in a 100 mm tissue culture dish and transduced 2times (4-6 hours each transduction) with freshly produced lentivirussupernatants. 200,000 transduced HUVECs were seeded on gelatin coated100-mm dish, cultured in HCM, and treated with 5 μM PS48, 0.25 mM NaBand 0.5 μM A-83-01 for 2 weeks, followed by changing half volume ofmedia to hESCM and supplementing with 5 μM PS48, 0.25 mM NaB and 0.5 μMA-83-01 for another 2 weeks. Then cell culture media were changed tohESCM and supplemented with 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01 and0.5 μM PD0325901 for additional 1-2 weeks. The iPSC colonies stainedpositive by Alexa Fluor 555 Mouse anti-Human TRA-1-81 antibody werepicked up for expansion on feeder cells in hESCM and cultured routinely.The culture was split by Accutase and treated with 1 μM Thiazovivin inthe first day after splitting.

Reprogramming of HUVECs Using Various Metabolism Modulating Compounds

HUVECs were cultured in a 100-mm tissue culture dish and transduced 2times (4-6 hours each transduction) with freshly produced lentivirussupernatants containing four reprogramming factors (Klf, Sox, Myc, andOct). About 20,000 transduced HUVECs were seeded on gelatin coated6-well plate, cultured in HCM, and treated with a metabolism modulatingcompound for 2 weeks. Then cell culture media were changed to hESCM andsupplemented with a metabolism modulating compound for additional 1-2weeks. The number of iPSC colonies stained positive by Alexa Fluor 555Mouse anti-Human TRA-1-81 antibody was counted. Various metabolismmodulating compounds have been tested, including 10 mM Fructose2,6-bisphosphate (F2,6P), 10 mM Fructose 6-phosphate (F6P), 10 μM6-aminonicotinamide (6-AN), 10 μM oxalate (OA), 1 μM 2,4-dinitrophenol(DNP), 1 μM N-oxalylglycine (NOG), 1 μM Quercetin (QC), 10 μM2-Hydroxyglutaric acid (2-HA), or 10 μM nicotinic acid (NA).

In Vitro Differentiation

The in vitro differentiation of hiPSCs was carried out by the standardembryoid body (EB) method. Briefly, the hiPSCs were dissociated byAccutase (Millipore), cultured in ultra-low attachment 6-well plate foreight days and then transferred to Matrigel-coated 6-well plate indifferentiation medium. The cells were fixed for immunocytochemicalanalysis or harvested for RT-PCR tests eight days later. Differentiationmedium: DMEM/F12, 10% FBS, 1% Glutamax, 1% Non-essential amino acids, 1%penicillin/streptomycin, 0.1 mM (3-mercaptoethanol.

Alkaline Phosphatase Staining and Immunocytochemistry Assay

Alkaline Phosphatase staining was performed according to themanufacturer's protocol using the Alkaline Phosphatase Detection Kit(Stemgent). Standard immunocytochemistry assay was carried out aspreviously reported (Li, W. et al., Stem Cells 27:2992-3000 (2009)).Primary antibodies used can be found in the Table 2. Secondaryantibodies were Alexa Fluor 488 donkey anti-mouse or anti-rabbit IgG(1:1000) (Invitrogen). Nuclei were visualized by DAPI (Sigma-Aldrich)staining. Images were captured using a Nikon Eclipse TE2000-Umicroscope.

Gene Expression Analysis by RT-PCR and qRT-PCR

For RT-PCR and qRT-PCR analysis, total RNA was extracted from humaniPSCs using the RNeasy Plus Mini Kit in combination with QIAshredder(Qiagen). First strand reverse transcription was performed with 2 μg RNAusing iScript™ cDNA Synthesis Kit (BioRad). The expression ofpluripotency markers was analyzed by RT-PCR using Platinum PCR SuperMix(Invitrogen). The expression of lineage specific markers afterdifferentiation was analyzed by qRT-PCR using iQ SYBR Green Supermix(Bio-Rad). The primers can be found in the Table 1.

Microarray Analysis

The Human Ref-8 v3 expression Beadchip (Illumina, Calif., USA) was usedfor microarray hybridizations to examine the global gene expression ofNHEKs, hiPSC and hES cells. Biotin-16-UTP-labeled cRNA was synthesizedfrom 500 ng total RNA with the Illumina TotalPrep RNA amplification kit(Ambion AMIL1791, Foster City, Calif., USA). The hybridization mixcontaining 750 ng of labeled amplified cRNA was prepared according tothe Illumina BeadStation 500× System Manual (Illumina, San Diego,Calif., USA) using the supplied reagents and GE HealthcareStreptavidin-Cy3 staining solution. Hybridization to the Illumina HumanRef-8_v3 expression Beadchip was for 18 h at 55° C. on a BeadChip HybWheel. The array was scanned using the Illumina BeadArray Reader. Allsamples were prepared in two biological replicates. Processing andanalysis of the microarray data were performed with the IlluminaBeadStudio software. The data were subtracted for background andnormalized using the rank invariant option.

Bisulfite Genomic Sequencing

Genomic DNAs were isolated using the Non Organic DNA Isolation Kit(Millipore) and then treated with the EZ DNA Methylation-Gold Kit (ZymoResearch Corp., Orange, Calif.). The treated DNAs were then used astemplates to amplify sequences of interest. Primers used for OCT4 andNANOG promoter fragment amplification are indicated in Table 1. Theresulting fragments were cloned using the TOPO TA Cloning Kit forsequencing (Invitrogen) and sequenced.

Genotyping of hiPSCs

Genotyping of hiPSC lines was performed using RT-PCR of genomic DNA withspecific primers (Table 1; Yu, J. et al., Science 318:1917-1920 (2007)and Li, W. et al., Stem Cells 27:2992-3000 (2009)).

Teratoma Formation

The hiPSC lines were harvested by using 0.05 Trypsin-EDTA. Five millioncells were injected under the kidney capsule of SCID mice (n=3). After4-6 weeks, well developed teratomas were harvested, fixed and thenhistologically analyzed at TSRI histology core facility.

TABLE 1 Primers used Gene Forward (SEQ ID NO:) Reverse (SEQ ID NO:)For RT-PCR Endo-OCT4 AGTTTGTGCCAGGGTTTTTG (12) ACTTCACCTTCCCTCCAACC (13)Endo-SOX2 CAAAAATGGCCATGCAGGTT (14) AGTTGGGATCGAACAAAAGCTATT (15) Endo-TTTGGAAGCTGCTGGGGAAG (16) GATGGGAGGAGGGGAGAGGA (17) NANOG Endo-KLF4ACGATCGTGGCCCCGGAAAAGGACC (18) GATTGTAGTGCTTTCTGGCTGGGCTCC (19)Endo-cMYC GCGTCCTGGGAAGGGAGATCCGGAGC TTGAGGGGCATCGTCGCGGGAGGCTG (20)(21) REX1 CAGATCCTAAACAGCTCGCAGAAT (22) GCGTACGCAAATTAAAGTCCAGA (23)UTF1 CCGTCGCTGAACACCGCCCTGCTG (24) CGCGCTGCCCAGAATGAAGCCCAC (25) TDGF2CTGCTGCCTGAATGGGGGAACCTGC (26) GCCACGAGGTGCTCATCCATCACAAGG (27) FGF4CTACAACGCCTACGAGTCCTACA (28) GTTGCACCAGAAAAGTCAGAGTTG (29) Exo-OCT4TGTCTCCGTCACCACTCTGG (30) ATGCATGCGGATCCTTCG (31) PAX6TGTCCAACGGATGTGAGT (32) TTTCCCAAGCAAAGATGGAC (33) βIIICAACAGCACGGCCATCCAGG (34) CTTGGGGCCCTGGGCCTCCGA (35) TUBULIN FOXF1AAAGGAGCCACGAAGCAAGC (36) AGGCTGAAGCGAAGGAAGAGG (37) HAND1TCCCTTTTCCGCTTGCTCTC (38) CATCGCCTACCTGATGGACG (39) AFPAGCAGCTTGGTGGTGGATGA (40) CCTGAGCTTGGCACAGATCCT (41) GATA6TGTGCGTTCATGGAGAAGATCA (42) TTTGATAAGAGACCTCATGAACCGACT (43) GAPDHGTGGACCTGACCTGCCGTCT (44) GGAGGAGTGGGTGTCGCTGT (45)For bisulfite-sequencing OCT4-1 TTAGGAAAATGGGTAGTAGGGATTT (46)TACCCAAAAAACAAATAAATTATAAAACC T (47) OCT4-2GGATGTTATTAAGATGAAGATAGTTGG (48) CCTAAACTCCCCTTCAAAATCTATT (49) NANOGGAGTTAAAGAGTTTTGTTTTTAAAAATTAT TCCCAAATCTAATAATTTATCATATCTTTC (50) (51)For genotyping OCT4-Int CAGTGCCCGAAACCCACAC (52)AGAGGAACTGCTTCCTTCACGACA (53) SOX2-Int TACCTCTTCCTCCCACTCCA (54)AGAGGAACTGCTTCCTTCACGACA (55) KLF4-Int CACCTTGCCTTACACATGAAGAGG (56)CGTAGAATCGAGACCGAGGAGA (57)

TABLE 2 Primary antibodies applied Antibody Species Dilution VendorAnti-OCT4 (1) Mouse 1:500 Santa Cruz Biotechnology Anti-OCT4 (2) Rabbit1:500 Stemgent Anti-SOX2 Rabbit  1:1000 Chemicon Anti-NANOG Rabbit 1:500Abcam Anti-SSEA4 Mouse 1:500 Stemgent Anti-TRA-1-81 Mouse 1:500 StemgentTUJ1 (Anti-βIII Mouse  1:3000 Covance Research Products TUBULIN)Anti-SMA Mouse 1:500 Sigma Anti-AFP Mouse 1:500 Sigma

TABLE 3 Summary of reprogramming experiments TRA-1-81 Induction Experi-positive Donor Cells factors Chemicals ments colonies NHEKs OCT4 + DMSO#1 17 (lot number: KLF4 + #2 20 0000087940) SOX2 + #3 23 MYC A83 + PD #172 #2 104 #3 91 OCT4 + DMSO #1 2 KLF4 + #2 3 SOX2 #3 8 A83 + PD #1 26 #235 #3 44 OCT4 + A83 + PD #1 1 KLF4 #2 2 #3 0 A83 + PS48 + PD #1 15 #2 18#3 5 A83 + VPA + PD #1 6 #2 0 #3 3 A83 + NaB + PD #1 20 #2 17 #3 18A83 + PS48 + NaB + #1 21 PD #2 30 #3 27 OCT4 A83 + PS48 + NaB + #1 4 PD#2 0 #3 3 NHEKs OCT4 A83 + PS48 + NaB + #1 2 (lot number: PD #2 32F0661) #3 0 AHEKs OCT4 A83 + PS48 + NaB + #1 3 PD + Par + CHIR #2 2HUVECs OCT4 A83 + PS48 + NaB + #1 4 PD #2 7 #3 4 HUVECs OCT4 A83 +PS48 + NaB + #1 23 PD + Par + CHIR #2 17 AFDCs OCT4 A83 + PS48 + NaB +#1 5 PD + Par + CHIR #2 11

NHEKs, Neonatal Human Epidermal Keratinocytes; HUVECs, Human UmbilicalVein Endothelial Cells; AHEKs, Adult Human Epidermal Keratinocytes;AFDCs, Amniotic Fluid Derived Cells. Chemical concentration used: PD,0.5 μM PD0325901; A83, 0.5 μM A-83-01; PS48, 5 μM PS48; VPA, 0.5 mMValproic acid; NaB, 0.25 mM Sodium butyrate; Par, 2 μM Parnate; CHIR, 3μM CHIR99021. For four-factor or three-factor induced reprogramming,NHEKs were seeded at a density of 100,000 transduced cells per 10 cmdish and positive colonies were counted four weeks later; For two-factorinduced reprogramming, NHEKs were seeded at a density of 100,000transduced cells per 10 cm dish and positive colonies were counted sixweeks later; and for one-factor induced reprogramming, NHEKs and AHEKswere seeded at a density of 1,000,000 transduced cells per 10 cm dishand positive colonies were counted eight weeks later. HUVECs and AFDCswere seeded at a density of 200,000 transduced cells per 10 cm dish andpositive colonies were counted six weeks later.

TABLE 4 Characterization of established human iPSC cell lines InductionCell Marker RT-PCR EB Teratoma hiPSC clone factors source expressiontest differentiation test hiPSC-OK#1 OCT4 + KLF4 NHEKs ✓ ✓ ✓ ✓hiPSC-OK#3 ✓ ✓ ✓ hiPSC-O#1 OCT4 NHEKs ✓ ✓ ✓ ✓ hiPSC-O#3 ✓ ✓ ✓ hiPSC-O#4✓ hiPSC-O#5 ✓ 2 more lines hiPSC-O#21 OCT4 HUVECs ✓ ✓ ✓ ✓ hiPSC-O#22 ✓hiPSC-O#26 ✓ ✓ ✓ hiPSC-O#31 ✓ ✓ ✓ ✓ 7 more lines hiPSC-O#52 OCT4 AHEKs ✓✓ hiPSC-O#57 ✓ hiPSC-O#63 OCT4 AFDCs ✓ ✓ hiPSC-O#65 ✓

Those cell lines characterized were long-term expanded for over 20passages under conventional hESC culture condition and furthercharacterized for marker expression and pluripotency; while other celllines established were stored at passage 5 or 6. Blank entries indicate“not determined.”

TABLE 5 DNA fingerprint analysis on Oct4-induced iPSCs and parental celllines Genomic hiPSC- hiPSC- loci NHEK (pooled) O#1 HUVEC O#21 AmelogeninX, Y X, Y X X vWA 11, 15, 17, 18, 19 15, 18 15; 16 15; 16 D8S1179 10,13, 16  13, 10; 13 10; 13 TPOX 8, 9, 11, 12  8 8 8 FGA 19, 22, 23, 2419, 22 24; 27 24; 27 D3S1358 13, 14, 15, 17 17 14; 16 14; 16 THO1 6, 7,9, 9.3 7, 9 6 6 D21S11 24.2, 29, 30.2, 35 24.2, 29     28; 30.2   28;30.2 D18S51 13, 14, 16, 17, 18, 19 13, 17 13; 18 13; 18 Penta E 5, 8,13, 14, 19 13, 19 12  12  D5S818 8, 11, 12, 13 11, 13 12; 13 12; 13D13S317 8, 9, 11, 12, 13  9, 12 11; 14 11; 14 D7S820 8, 9, 10, 11  9, 1011  11  D16S539 9, 10, 11, 12, 13  9, 13  9; 11  9; 11 CSF1PO 10, 11, 1211, 12 11; 12 11; 12 Penta D 2.2, 10, 12 10 12; 13 12; 13

Fifteen polymorphic short tandem repeat (STR) DNA loci and the sexchromosome marker amelogenin were investigated.

Example 2: Reprogramming of Human Umbilical Vein Endothelial Cells

We tested the effects of the combination of a HDAC inhibitor, a PDK1activator, a TGFβ receptor inhibitor, and a MEK inhibitor on HUVECs thatwere lentivirally transduced with Oct4 alone for their effects onreprogramming kinetics and efficiency.

Methods

Human Umbilical Vein Endothelial Cells (HUVECs, Millipore) weremaintained in EndoGRO-VEGF Complete Medium (HCM, CHEMICON). HUVECs werecultured in a 100 mm tissue culture dish and transduced 2 times (4-6hours/time) with freshly produced lentivirus supernatants. Then 200,000transduced HUVECs were seeded on gelatin coated 100-mm dish and culturedin HCM and treated with PDK1 activator PS48 (5 μM), HDAC inhibitor NaB(0.25 mM), and TGFβ receptor inhibitor A-83-01(0.5 μM) for 2 weeks,followed by changing half volume of media to hESCM and supplementingwith PDK1 activator PS48 (5 μM), HDAC inhibitor NaB (0.25 mM), and TGFβreceptor inhibitor A-83-01(0.5 μM) for another 2 weeks. Then cellculture media were changed to hESCM and supplemented with PDK1 activatorPS48 (5 μM), HDAC inhibitor NaB (0.25 mM), and TGFβ receptor inhibitorA-83-01(0.5 μM) and MEK inhibitor PD0325901 (0.5 μM) for additional 2weeks. The iPSC colonies were stained positive by Alexa Fluor 555 Mouseanti-Human TRA-1-81 antibody (BD Pharmingen). hESCM: DMEM/F12, 15%Knockout serum replacement, 1% Glutamax, 1% Non-essential amino acids,1% penicillin/streptomycin, 0.1 mM β-mercaptoethanol and 10 ng/ml bFGF.

Results

For HUVECs transduced with Oct4 alone, we tested the effects of thecombination of a HDAC inhibitor, a PDK1 activator, a TGFβ receptorinhibitor, a MEK inhibitor on reprogramming efficiency. We found thattreatment with the combination of 5 μM PS48, 0.25 mM NaB, 0.5 μM A-83-01and 0.5 μM PD0325901 results in a ˜0.0015% reprogramming efficiency.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences,patents, and patent applications cited herein are hereby incorporated byreference.

What is claimed is:
 1. A composition comprising an induced mammalianpluripotent stem cell (iPSC) and a cell culture medium, wherein: (a) theiPSC comprises an exogenous polynucleotide encoding an Oct4 polypeptide;(b) the cell culture medium comprises: (i) a small molecule selectedfrom the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P(fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine),QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), andPDK1 activator (3′-phosphoinositide-dependent kinase-1), and (ii) one orboth of a TGFβ receptor/ALK5 inhibitor and a MEK inhibitor; and (c) theiPSC is reprogrammed from a non-pluripotent mammalian cell; whereincontacting the non-pluripotent mammalian cell with the small moleculeenhances reprogramming when compared to without the small molecule. 2.The composition of claim 1, wherein the PDK1 activator is: (a) anallosteric PDK1 activator; or (b)(Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (PS48),(Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (PS08),2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (12Z), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (13Z).
 3. Thecomposition of claim 1, wherein the iPSC further comprises one or moreof: (a) an exogenous polynucleotide encoding a Klf polypeptide; and (b)an exogenous polynucleotide encoding a Sox-2 polypeptide.
 4. Thecomposition of claim 1, wherein the non-pluripotent mammalian cell is(a) a human cell; or (b) a somatic cell, a progenitor cell, or a fullydifferentiated cell.
 5. The composition of claim 1, wherein the cellculture medium further comprises the TGFβ receptor/ALK5 inhibitor.
 6. Acomposition for use in manufacturing a mammalian pluripotent stem cellfor application in regenerative medicine, the composition comprising acell culture medium comprising a small molecule and one or both of aTGFβ receptor/ALK5 inhibitor and a MEK inhibitor, wherein (i) the smallmolecule is present in an amount that enhances reprogramming of anon-pluripotent mammalian cell comprising an exogenous polynucleotideencoding an Oct4 polypeptide and (ii) the small molecule is selectedfrom the group consisting of F2,6P (fructose 2,6-bisphosphate), F6P(fructose 6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine),QC (quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), or aPDK1 (3′-phosphoinositide-dependent kinase-1) activator.
 7. Thecomposition of claim 6, wherein the PDK1 activator is: (a) an allostericPDK1 activator; or (b) (Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid(PS48), (Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (PS08),2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio)acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (12Z), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (13Z).
 8. Thecomposition of claim 6, wherein the mammalian cell is (a) a human cell;or (b) a somatic cell, a progenitor cell, a fully differentiated cell, anon-pluripotent cell, or a pluripotent cell.
 9. A kit comprising amixture of (A) a cell culture medium, (B) a MEK inhibitor, and (C) asmall molecule in an amount that enhances reprogramming of anon-pluripotent mammalian cell when used in a method of obtaining aninduced mammalian pluripotent stem cell (iPSC) comprising: (a)introducing into a non-pluripotent mammalian cell a polynucleotideencoding an Oct4 polypeptide; (b) contacting the non-pluripotentmammalian cell with (i) the small molecule, wherein the small moleculeenhancing reprogramming is selected from the group consisting of F2,6P(fructose 2,6-bisphosphate), F6P (fructose 6-phosphate), DNP(2,4-dinitrophenol), NOG (N-oxalylglycine), QC (quercetin), 2-HA(2-hydroxyglutaric acid), NA (nicotinic acid), and PDK1 activator(3′-phosphoinositide-dependent kinase-1), and (ii) the MEK inhibitor;and (c) culturing the contacted non-pluripotent mammalian cell of (b) inthe culture medium, thereby reprogramming the non-pluripotent mammaliancell to produce an induced mammalian pluripotent stem cell; whereincontacting the non-pluripotent mammalian cell with the small moleculeenhances reprogramming when compared to without the small molecule. 10.The kit of claim 9, further comprising a non-pluripotent mammalian cell.11. The kit of claim 9, further comprising at least one exogenoustranscription factor comprising Oct4, and optionally one or both of Sox2and Klf; wherein the at least one exogenous transcription factor is apolypeptide, or a polynucleotide encoding the polypeptide.
 12. The kitof claim 9, wherein: (a) the PDK1 activator is an allosteric PDK1activator; or (b) the PDK1 activator is(Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (PS48),(Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (PS08),2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio) acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (12Z), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (13Z); or (c) the MEKinhibitor is PD0325901.
 13. The kit of claim 9, further comprising oneor more of a TGFβ receptor/ALK5 inhibitor, a glycogen synthase kinase 3(GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor and whereinsaid method further comprises contacting the non-pluripotent mammaliancell with said one or more of a TGFβ receptor/ALK5 inhibitor, a glycogensynthase kinase 3 (GSK3) inhibitor, and a histone deacetylase (HDAC)inhibitor.
 14. The kit of claim 13, wherein (a) the TGFβ receptor/ALK5inhibitor is A-83-01; or (b) the HDAC inhibitor is sodium butyrate (NaB)or valproic acid (VPA).
 15. The kit of claim 9, wherein thenon-pluripotent mammalian cell is (a) a human cell; or (b) a somaticcell, a progenitor cell, or a fully differentiated cell.
 16. A method ofmaking an induced mammalian pluripotent stem cell (iPSC), the methodcomprising: (a) introducing into a non-pluripotent mammalian cell apolynucleotide encoding an Oct4 polypeptide; (b) contacting thenon-pluripotent mammalian cell with (i) a small molecule selected fromthe group consisting of F2,6P (fructose 2,6-bisphosphate), F6P (fructose6-phosphate), DNP (2,4-dinitrophenol), NOG (N-oxalylglycine), QC(quercetin), 2-HA (2-hydroxyglutaric acid), NA (nicotinic acid), andPDK1 activator (3′-phosphoinositide-dependent kinase-1), and (ii) one orboth of a TGFβ receptor/ALK5 inhibitor and a MEK inhibitor; and (c)culturing the contacted non-pluripotent mammalian cell of (b), therebyreprogramming the non-pluripotent mammalian cell to produce an inducedmammalian pluripotent stem cell; wherein contacting the non-pluripotentmammalian cell with the small molecule enhances reprogramming whencompared to without the small molecule; and further wherein (iii) theMEK inhibitor is PD0325901, or (iv) the method further comprisescontacting the non-pluripotent mammalian cell with one or more of aglycogen synthase kinase 3 (GSK3) inhibitor, and a histone deacetylase(HDAC) inhibitor.
 17. The method of claim 16, wherein: (a) the PDK1activator is an allosteric PDK1 activator; or (b) the PDK1 activator is(Z)-5-(4-Chlorophenyl)-3-phenylpent-2-enoic acid (PS48),(Z)-5-(4-Bromo-2-fluorophenyl)-3-phenylpent-2-enoic acid (PS08),2-(3-(4-Chlorophenyl)-3-oxo-1-phenylpropylthio) acetic acid,(Z)-5-(Napthalen-2-yl)-3-phenylpent-2-enoic acid (12Z), or(Z)-5-(1H-Indol-3-yl)-3-phenylpent-2-enoic acid (13Z); or (c) the MEKinhibitor is PD0325901.
 18. The method of claim 16, wherein said methodfurther comprises contacting the non-pluripotent mammalian cell with oneor more of a TGFβ receptor/ALK5 inhibitor, a glycogen synthase kinase 3(GSK3) inhibitor, and a histone deacetylase (HDAC) inhibitor.
 19. Themethod of claim 18, wherein (a) the TGFβ receptor/ALK5 inhibitor isA-83-01; or (b) the HDAC inhibitor is sodium butyrate (NaB) or valproicacid (VPA).
 20. The method of claim 16, wherein the non-pluripotentmammalian cell is (a) a human cell; or (b) a somatic cell, a progenitorcell, or a fully differentiated cell.